Antiviral conjugate

Abstract

The present invention relates to an antiviral conjugate. Specifically, it relates to a conjugate represented by formula (I), where E comprises a protein or a polypeptide; L is a linker that covalently links E to D; and D is selected independently of the structure represented by formula (A). The antiviral conjugate can effectively treat related diseases, such as viral infections.

Description

An antiviral conjugate Technical Field

[0001] The present disclosure belongs to the field of medicine, and specifically relates to an antiviral conjugate. Background Art

[0002] Influenza viruses, including both human and animal influenza viruses, can cause infection and illness in a variety of animals, including humans, poultry, pigs, horses, and bats. Influenza viruses are divided into four types: A, B, C, and D. Influenza A viruses spread rapidly, easily spreading from person to person and even across species. Due to the high antigenic variation of influenza viruses, they have repeatedly caused global pandemics. These pandemics bring devastating consequences to human health and the global economy.

[0003] Currently, clinically used anti-influenza drugs are primarily neuraminidase (NA) inhibitors (such as oseltamivir) and M2 ion channel protein inhibitors (such as amantadine and rimantadine). Early symptomatic use of these drugs can shorten the course of influenza and alleviate symptoms, but they also have drawbacks. Drug resistance has become a significant issue in the use of anti-influenza drugs that cannot be ignored.

[0004] Studies have shown that drug-resistant influenza A (H3N2) viruses have emerged globally since 2003, and in 2007, oseltamivir-resistant seasonal influenza A (H1N1) viruses emerged worldwide. The incidence of amantadine-resistant influenza A (H3N2) viruses has reached nearly 100%, while the incidence of amantadine-resistant influenza A (H1N1) viruses has reached 15.5%. Therefore, the development of new anti-influenza drugs is urgently needed.

[0005] Several drugs for inhibiting influenza virus growth have been disclosed, including novel conjugates. For example, WO2020051498 discloses a series of antiviral drug conjugates. Summary of the Invention

[0006] The purpose of the present disclosure is to provide a new antiviral conjugate.

[0007] In one aspect, the present disclosure provides a conjugate represented by formula (I),

[0008] in,

[0009] E contains a protein or polypeptide;

[0010] L is a linker that covalently links E to D;

[0011] m is selected from 3, 4, 5, 6, 7, 8, 9 and 10;

[0012] n is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number between any two numbers);

[0013] D is independently selected from the structure represented by formula (A),

[0014] R1 is selected from -OH, -NH2 and -NHC(=NH)NHR5;

[0015] R2 is selected from -CO2H, -P(=O)(OH)2 and -SO3H;

[0016] R3 is -O- or -S-;

[0017] R4 is selected from -COCH3, -COCF3 and -SO2CH3;

[0018] R5 is selected from hydrogen, hydroxy, mercapto, nitro, cyano, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k 、C1-C 10 Alkyl, C1-C 10 Alkoxy, C2-C 10 Alkenyl and C2-C 10 Alkynyl, wherein the alkyl, alkoxy, alkenyl and alkynyl are optionally selected from C1-C6 alkyl, halogen, hydroxy, mercapto, -NR i R j , oxo, thio, -C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k , nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl;

[0019] R i 、R j Each independently selected from a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group and a C1-C6 alkoxy group;

[0020] R k independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl, -NR i R j wherein the alkyl, alkoxy, and haloalkyl are optionally selected from C1-C6 alkyl, halogen, hydroxyl, mercapto, -NR i R j , oxo, thioxo, carboxyl, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl and 5- to 10-membered heteroaryl.

[0021] In some embodiments, R5 is selected from hydrogen, hydroxy, amino, and -C(O)R k , where R k Selected from hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy and hydroxy, wherein the alkyl and alkoxy are optionally substituted by one or more substituents selected from halogen, hydroxy, oxo, thioxo, carboxyl, C1-C6 alkoxy, C1-C6 alkylthio, 6 to 10 membered aryl and 5 to 10 membered heteroaryl.

[0022] In some embodiments, D is selected from the structure shown in formula (A-1),

[0023] In some embodiments, m is 3 or 4.

[0024] In some embodiments, the linker comprises a backbone unit connected to D. The backbone unit may comprise a core portion L2 and branch portions L1 and L3. The branch portion L1 is used to connect E to the core portion. The branch portion L3 is used to connect D to the core portion. There may be multiple branches L3 connected to the core portion.

[0025] In some embodiments, the linker comprises a moiety having (—CH 2 (CH 2 ) x2 -Y1-) x1 A repeating unit structure of , wherein x1 is an integer from 0 to 100, x2 is an integer from 0 to 10, and Y1 is selected from O or CH2, such as O. In some embodiments, x2 is 1. In some embodiments, the linker includes a polyethylene glycol (PEG) structure.

[0026] In some embodiments, the core structure L2 is selected from

[0027] wherein y1 is an integer from 0 to 100, such as an integer from 0 to 30, y2 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2, and Y b1 is selected from O or CH2, such as O, p1, p2 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2, or

[0028] wherein z1 is an integer from 0 to 100, such as an integer from 0 to 30, z2 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2, and Y b2 is selected from O or CH2, such as O, z3 is an integer from 0 to 100, such as an integer from 0 to 30, z4 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2, Y b3 is selected from O or CH2, q1, q2, q3, q4 are each independently selected from an integer of 0-10, for example an integer of 0-6 or

[0029] The E or D can be connected to any nitrogen atom.

[0030] In some embodiments, the linker comprises a branched structure L1, which is a chemical structure fragment having one end covalently linked to E via a carbon atom and the other end optionally linked to the core portion of the linker via a repeating unit structure.

[0031] In some embodiments, molecules containing an azide group can be used to form a terminal group, wherein the azide group can undergo a cycloaddition with an alkyne to form a 1,2,3-triazole linkage. In some embodiments, molecules containing an alkynyl group can be used to form a terminal group, wherein the alkynyl group can undergo a cycloaddition with an azide to form a 1,2,3-triazole linkage. In some embodiments, molecules containing a maleimide group can be used to form a terminal group, wherein the maleimide group can react with cysteine ​​to form a CS linkage. In some embodiments, molecules containing one or more sulfonic acid groups can be used to form a terminal group, wherein the sulfonic acid group can form a sulfonamide linkage with the connecting nitrogen in the neuraminidase inhibitor. In some embodiments, molecules containing one or more isocyanate groups can be used to form a terminal group, wherein the isocyanate group can form a urea linkage with the connecting nitrogen in the neuraminidase inhibitor. In some embodiments, molecules containing one or more haloalkyl groups can be used to form a terminal group, wherein the haloalkyl group can form a covalent linkage with the neuraminidase inhibitor, such as a CN and CO linkage.

[0032] Covalent coupling of two or more components in a conjugate using end groups can be achieved using well-known organic chemical synthesis techniques and methods. Complementary functional groups on the two components can react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and olefin and tetrazine. Site-specific coupling with polypeptides (e.g., Fc monomers, Fc domains, Fc-binding peptides, albumin or albumin-binding domains, etc.) can be achieved using techniques known in the art.

[0033] In some embodiments, the polypeptide E and the small molecule D described in the present disclosure can be coupled by the following means: (a) thiourea linkage (i.e., -NH(C=S)NH-) to the lysine of E; (b) carbamate linkage (i.e., -NH(C=O)-O) to the lysine of E; (c) amine linkage (i.e., -NHCH2) between lysine and E by reductive amination; (d) amide (i.e., -NH-(C=O)CH2) to the lysine of E; (e) cysteine-maleimide binding between the maleimide in the terminal group and the cysteine ​​of E; (f) cysteine-maleimide binding between the terminal group and the carbohydrate of E (e.g., Fc (e.g., an Fc monomer, an Fc domain, an Fc-binding peptide, a carbohydrate group of albumin or an albumin-binding domain); (g) a rebridging cysteine ​​linkage, wherein the terminal group is coupled to two cysteines of E; (h) an oxime linkage between the terminal group and a carbohydrate of E (e.g., an Fc monomer, an Fc domain, an Fc-binding peptide, a carbohydrate group of albumin or an albumin-binding domain); (i) an oxime linkage between the terminal group and an amino acid residue of E; (j) an azido linkage between the terminal group and E; (k) direct acylation of the terminal group to E; or (l) a thioether linkage between the terminal group and E.

[0034] In some embodiments, the PEG linker is functionalized by an active ester, such as a nitrophenyl ester or an N-hydroxysuccinimide ester, or a derivative thereof, such as a PEG linker (e.g., an azido-PEG2-PEG 40 -NHS ester) conjugated to E. In these cases, E-(PEG2-PEG 40 )-azide can be reacted via click conjugation to an intermediate with a terminal alkyne linker. During click conjugation, the catalyzed azide (e.g., Fc-(PEG2-PEG 40 )-azide) reacts with the terminal alkyne group of the intermediate to form a 5-membered heteroatom ring.

[0035] In some embodiments, L1 may comprise a reactive group G linked directly or indirectly to E as follows x ,

[0036] g are each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl.

[0037] In some embodiments, L1 forms an E-L1- structure with E, and L1 is selected from -G a1 -(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -, wherein xa1, xa3 are each independently selected from an integer of 0 to 100, such as an integer of 0 to 30, xa2, xa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2, wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, Y a1 、Y a2 Each independently selected from O or CH2, such as O, G a1 Selected from group G x , G a2 , G a3 Each independently selected from the group G x Or does not exist.

[0038] In some embodiments, L1 is selected from -G a1 -(CH2) wa1 -[O-CH2CH2] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[O-CH2CH2] xa3 -(CH2) wa4 -G a3 -.

[0039] In some embodiments, the linker comprises an amino acid unit, for example, comprising 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, n-methyl-valine, (q is an integer from 1 to 6) peptide residues composed of amino acids, exemplary amino acid units include but are not limited to valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala) and glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit) and glycine-glycine-glycine (Gly-Gly-Gly),

[0040] In some embodiments, the branch portion L3 comprises a terminal group G connected to D c1 , forming -G c1 -D structure. The terminal group G c1 It can be selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl, for example from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2.

[0041] In some embodiments, L3 is -(CH2) wc1 -[Y c1 -CH2(CH2) xc2 ] xc1 -(CH2) wc2 -G c1 -,

[0042] wherein xc1 is each independently an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0043] xc2 are each independently an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0044] Y c1 are each independently selected from O or CH2, such as O,

[0045] wc1 and wc2 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 0, 1, 2 or 3,

[0046] G c1Each is independently selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl, for example, from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2;

[0047] m is 3 or 4.

[0048] In some embodiments, L3 is -(CH2) wc1 -[O-CH2CH2] xc1 -(CH2) wc2 -G c1 -.

[0049] In some embodiments, xc1 is each independently an integer from 2 to 30, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0050] In some embodiments, the number of atoms in the L3 main chain is greater than 7, for example greater than 8, for example greater than 9, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therebetween, for example 8-30, or 8-20.

[0051] The number of atoms in the L3 main chain can be understood as the number of atoms in the -(CH2) group. wc1 To Group-G c1 -The number of atoms in the straight chain between. For example, if L3 is The main chain is The number of main chain atoms is 7; if L3 is The main chain is The number of main chain atoms is 16.

[0052] In some embodiments, -L(-D) m -L1-L2(-L3-D) m ,in,

[0053] L1 is a portion connected to E, and L1 is selected from -G a1 -(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2)xa4 ] xa3 -(CH2) wa4 -G a3 -,in,

[0054] xa1 and xa3 are each independently selected from an integer from 0 to 100, for example an integer from 0 to 30,

[0055] xa2 and xa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2,

[0056] Wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 0, 1, 2,

[0057] Y a1 、Y a2 are each independently selected from O or CH2, such as O,

[0058] G a1 Selected from group G x , G a2 , G a3 Each independently selected from the group G x Or does not exist; G x As mentioned before;

[0059] L2 is selected from

[0060] wherein y1 is an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0061] y2 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0062] Y b1 Selected from O or CH2, such as O,

[0063] p1 and p2 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2; or

[0064] wherein z1 is an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0065] z2 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0066] Y b2 Selected from O or CH2, such as O,

[0067] z3 is an integer from 0 to 100, for example an integer from 0 to 30, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0068] z4 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0069] Y b3 Selected from O or CH2, preferably O,

[0070] q1, q2, q3, q4 are each independently selected from an integer of 0-10, such as an integer of 0-6; or

[0071] L3 is -(CH2) wc1 -[Y c1 -CH2(CH2) xc2 ] xc1 -(CH2) wc2 -G c1 -,

[0072] wherein xc1 is each independently an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0073] xc2 are each independently an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0074] Y c1 are each independently selected from O or CH2, such as O,

[0075] wc1 and wc2 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 0, 1, 2 or 3,

[0076] G c1 Each is independently selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl, for example, from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2;

[0077] m is 3 or 4.

[0078] In some embodiments, xc1 is each independently an integer from 2 to 30, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therebetween, for example, 2-20, etc.

[0079] In some embodiments, the number of atoms in the L3 backbone is greater than 7, for example greater than 8, for example greater than 9, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therebetween, for example 8 to 30, or 8 to 20. In some embodiments, n is any number between 1 and 15, for example, any number between 2 and 12. n can be an integer or a decimal.

[0080] In some embodiments, E comprises a half-life extending domain; for example, said E is selected from an Fc domain, albumin, or an albumin binding domain.

[0081] In some embodiments, the Fc domain is a monomer or dimer. In some embodiments, the Fc domain is an Fc domain monomer that includes C H 2 Antibody constant domain and C H 3 antibody constant domain. In some embodiments, the Fc domain monomer may have an immunoglobulin antibody isotype IgG, IgE, IgM, IgA or IgD. The Fc domain monomer may also have any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3 or IgG4). The Fc domain monomer may have any immunoglobulin antibody allotype (e.g., IGHG1*01 (i.e., G1m(za)), IGHG1*07 (i.e., G1m(zax)), IGHG1*04 (i.e., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (i.e., G1m(fa)), IGHG2*01, IGHG2*06, IGHG2*02, IGHG3*01, IGHG3*05, IGHG In some embodiments, the Fc domain monomers may be derived from any species, such as human, murine, or mouse. A dimerized Fc domain monomer can bind to the Fc domain of an Fc receptor, which is a receptor located on the surface of a leukocyte. In some embodiments, the Fc domain includes a hinge domain; in some embodiments, the Fc domain does not include a hinge domain.

[0082] In some embodiments, the Fc domains described herein include H 3 Interactions between antibody constant domains Two Fc domain monomers that dimerize, and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. In some embodiments, the two Fc domain monomers may be the same or different. The Fc domain comprises a minimal structure capable of binding to an Fc receptor, such as an Fc-gamma receptor (i.e., Fcgamma receptor (FcγR)), an Fc-alpha receptor (i.e., Fcα receptor (FcαR)), an Fc-epsilon receptor (i.e., Fcε receptor (FcεR)), and / or a neonatal Fc receptor (FcRn). In some embodiments, the Fc domains described herein bind to an Fcgamma receptor (e.g., FcRn, FcgammaRI (CD64), FcgammaRIIa (CD32), FcgammaRIIb (CD32), FcgammaRIIIa (CD16a), FcgammaRIIIb (CD16b)) and / or FcgammaRIV and / or a neonatal Fc receptor (FcRn).

[0083] In some embodiments, the Fc domain comprises a native Fc region, or an Fc region variant having one or more alterations relative to a native Fc region. Alterations may include amino acid substitutions, additions and / or deletions, attachment of additional moieties, and / or alterations in native glycans. In some embodiments, the Fc domain also encompasses a single-chain Fc region in which the constituent Fc domains are linked together by a linker moiety.

[0084] In some embodiments, the Fc domain described herein is an aglycosylated Fc domain (e.g., an Fc domain that maintains the ability to bind to an Fc receptor (e.g., FcRn). For example, the Fc domain is an aglycosylated IgG1 variant that maintains the ability to bind to an Fc receptor (e.g., IgG1 having amino acid substitutions at N297 and / or T299 of the glycosylation motif). Exemplary aglycosylated Fc domains and methods for preparing aglycosylated Fc domains are known in the art.

[0085] The Fc domain described in the present disclosure may be selected from an antibody.

[0086] Antibodies include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH domains (VH) or VL domains (VL). Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heterologous conjugates, antibody-drug conjugates, single domain antibodies (sdAbs), monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelid antibodies, affibody molecules, VHH fragments, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), and antigen-binding fragments of any of the above antibodies.

[0087] In some embodiments, the antibody is a human, mouse, camelid (e.g., llama, alpaca, or camel), goat, sheep, rabbit, chicken, guinea pig, hamster, horse, or rat antibody or antigen-binding fragment. In some embodiments, the antibody is IgG, IgA, IgD, IgE, or IgM. In some embodiments, the antigen-binding fragment includes Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above.

[0088] In some embodiments, the antibodies confer binding specificity for one or more targets (eg, antigens).

[0089] In some embodiments, the one or more targets (e.g., antigens) bound by the antibody are viral (e.g., influenza) proteins, such as neuraminidase or hemagglutinin. In some embodiments, the antibody or antibody fragment recognizes viral surface antigens. In some embodiments, the antibody targets hemagglutinin. Hemagglutinin targeting antibodies include monoclonal antibodies, such as CR6261, CR8020, MEDI8852, MHAA4549A, and VIS410. In some embodiments, the antibody is a broadly neutralizing antibody or antigen-binding fragment targeting influenza hemagglutinin. In some embodiments, the antibody targets viral matrix proteins (e.g., matrix 2 protein). TCN032 is a matrix 2 protein targeting monoclonal antibody. In some embodiments, the antibody targets human serum albumin.

[0090] In some embodiments, the antibody comprises one or more single domain antibodies (sdAbs). In some embodiments, the Fc domain-containing composition is an antibody or antibody fragment comprising an sdAb with influenza A reactivity, such as an sdAb conjugated to influenza A hemagglutinin. In some embodiments, the antibody comprises an sdAb with influenza B reactivity, such as an sdAb conjugated to influenza B hemagglutinin.

[0091] In some embodiments, the antibody is a multidomain antibody (MDAb) or a multidomain antibody fragment comprising two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten or more) sdAbs. In some embodiments, the MDAb or fragment thereof comprises one or more sdAbs conjugated to an influenza A hemagglutinin and one or more sdAbs conjugated to an influenza B hemagglutinin.

[0092] In some embodiments, the Fc domain has enhanced effector function, e.g., the effector function is selected from the group consisting of: C1q binding and complement-dependent cytotoxicity, Fc receptor (e.g., FcγR) binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0093] In some embodiments, the Fc domain may be the following sequence:

[0094] (I) a sequence as set forth in SEQ ID NO: 1 or at least 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 1; or

[0095] (II) a sequence as set forth in SEQ ID NO: 2, or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 2;

[0096] (III) a sequence as set forth in SEQ ID NO: 3, or a sequence that is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 3; or

[0097] (IV) a sequence as set forth in SEQ ID NO: 4, or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 4; or

[0098] (V) a sequence as set forth in SEQ ID NO: 5 or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 2;

[0099] (VI) a sequence as set forth in SEQ ID NO:6, or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:3; or

[0100] (VII) a sequence as set forth in SEQ ID NO: 7 or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 4; or

[0101] (VIII) a sequence as set forth in SEQ ID NO:8, or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:8.

[0102] In some embodiments, the Fc domain is an antibody. Exemplarily, the antibody comprises a heavy chain and a light chain. Exemplarily, the heavy chain comprises a sequence as set forth in SEQ ID NO: 9, or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 9; and the light chain comprises a sequence as set forth in SEQ ID NO: 10, or at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 10.

[0103] All mutations are numbered according to the EU numbering system, and the specific mutation sites are bold and underlined.

[0104] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E mutations

[0105] SEQ ID NO: 1

[0106] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E, H268C, L443C mutations

[0107] SEQ ID NO: 2

[0108] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E, K290C, L443C mutations

[0109] SEQ ID NO: 3

[0110] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E, L398C, L443C mutations

[0111] SEQ ID NO: 4

[0112] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E, T223Y, P329Y mutations

[0113] SEQ ID NO: 5

[0114] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E, P329Y mutations and GGGGY sequence added to the C-terminus

[0115] SEQ ID NO: 6

[0116] >Human IgG1 Fc fragment (Asn201-Gly446) with C220S, M252Y, S254T, T256E, T223Y mutations and GGGGY sequence added to the C-terminus

[0117] SEQ ID NO: 7

[0118] >Human IgG1 Fc fragment (Asn201-Gly446) with C220A, M252Y, S254T, T256E mutations

[0119] SEQ ID NO: 8

[0120] >hIgG ​​antibody heavy chain

[0121] SEQ ID NO: 9

[0122] >hIgG ​​antibody light chain

[0123] SEQ ID NO: 10

[0124] In some embodiments, the albumin is human albumin, such as human serum albumin.

[0125] In some embodiments, the albumin binding domain is a polypeptide that binds to albumin, or a domain that binds to albumin, such as an antibody or an antigen-binding fragment thereof (eg, VHH).

[0126] In some embodiments, the albumin or albumin binding domain is modified. For example, it is modified to contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8) solvent-exposed cysteine ​​(Cys) or lysine (Lys) residues. In some embodiments, the modification is an amino acid mutation (e.g., substitution). In some embodiments, the albumin or albumin binding domain is conjugated to a compound of the present disclosure (e.g., a neuraminidase inhibitor monomer or dimer) via a modified or naturally occurring Cys and / or Lys.

[0127] In some embodiments, a conjugate of the present disclosure may contain one or more (e.g., 2, 3, 4) proteins or polypeptides, which may be any of the Fc domains, albumin, or albumin-binding domains of the present disclosure. In some embodiments, a conjugate of the present disclosure may contain one or more (e.g., 2, 3, 4) identical proteins or polypeptides; in other embodiments, a conjugate of the present disclosure may contain multiple (e.g., 2, 3, 4) different proteins or polypeptides.

[0128] In some embodiments, the antiviral conjugate of the present disclosure is selected from:

[0129] L1 and E form an E-L1- structure, and L1 is selected from -G a1 -(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -, wherein xa1, xa3 are each independently selected from an integer of 0 to 100, such as an integer of 0 to 30, xa2, xa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2, wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, Y a1 、Ya2 Each independently selected from O or CH2, G a1 Selected from group G x , G a2 , G a3 Each independently selected from the group G x or does not exist;

[0130] G x Selected from:

[0131] g are each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl;

[0132] G c1 Selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2-, wherein R6 is selected from hydrogen and C1-C6 alkyl;

[0133] n is any value between 1 and 15.

[0134] In some embodiments, the antiviral conjugate of the present disclosure is selected from:

[0135] in,

[0136] xa5 are each independently selected from an integer between 2 and 8,

[0137] G a1 Each independently selected from

[0138] g are each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl;

[0139] n is any value between 1 and 15.

[0140] The present disclosure also provides a compound as shown below, L1'-L2(-L3-D) m ,

[0141] in,

[0142] L1' is selected from G x '-(CH2) wa1 -[Ya1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -,in,

[0143] xa1 and xa3 are each independently selected from an integer from 0 to 100, for example an integer from 0 to 30,

[0144] xa2 and xa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2,

[0145] Wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 0, 1, 2,

[0146] Y a1 、Y a2 are each independently selected from O or CH2, such as O,

[0147] G x 'Each independently selected from

[0148] G a2 , G a3 Each independently selected from:

[0149] or does not exist;

[0150] g are each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl;

[0151] Ring H is a 5- to 10-membered heteroaryl group, Can be selected from

[0152] For example

[0153] R h are each independently selected from halogen;

[0154] h is selected from 0, 1, 2, 3, 4, 5;

[0155] h1 is independently selected from 0, 1, and 2;

[0156] h2 are each independently selected from 0, 1, 2, 3;

[0157] h3 are each independently selected from 0, 1, 2, 3, 4;

[0158] h4 are each independently selected from 0, 1, 2, 3, 4, 5;

[0159] L2 is selected from

[0160] wherein y1 is an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0161] y2 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0162] Y b1 Selected from O or CH2, such as O,

[0163] p1 and p2 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 1 or 2; or

[0164] wherein z1 is an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0165] z2 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0166] Y b2 Selected from O or CH2, such as O,

[0167] z3 is an integer from 0 to 100, for example an integer from 0 to 30, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0168] z4 is an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0169] Y b3 Selected from O or CH2, such as O,

[0170] q1, q2, q3, q4 are each independently selected from an integer of 0-10, such as an integer of 0-6; or

[0171] L3 is -(CH2) wc1 -[Y c1 -CH2(CH2) xc2 ] xc1 -(CH2) wc2 -G c1-,in,

[0172] xc1 are each independently an integer from 0 to 100, such as an integer from 0 to 30, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

[0173] xc2 are each independently an integer from 0 to 10, such as an integer from 0 to 6, such as 1 or 2,

[0174] Y c1 are each independently selected from O or CH2, such as O,

[0175] wc1 and wc2 are each independently selected from an integer of 0-10, such as an integer of 0-6, such as 0, 1, 2 or 3,

[0176] G c1 Each is independently selected from NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl, for example from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2;

[0177] D is

[0178] m is 3 or 4.

[0179] In some embodiments, xc1 is each independently an integer from 2 to 30, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therebetween, for example, 2-20, etc.

[0180] In some embodiments, the number of atoms in the L3 main chain is greater than 7, for example greater than 8, for example greater than 9, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any range therebetween, for example 8-30, or 8-20.

[0181] In some embodiments, the compound is selected from:

[0182] in,

[0183] xa6 are each independently selected from an integer between 0 and 8,

[0184] G x 'Each independently selected from

[0185] In some embodiments, the compound is selected from:

[0186] The present disclosure also provides a method for preparing the antiviral conjugate of the present disclosure, comprising the steps of coupling polypeptide E with small molecule D,

[0187] The polypeptide E and the small molecule D can be coupled by the following methods: including but not limited to (a) thiourea linkage (i.e., -NH(C=S)NH-) to the lysine of E; (b) carbamate linkage (i.e., -NH(C=O)-O) to the lysine of E; (c) amine linkage (i.e., -NHCH2) between lysine and E by reductive amination; (d) amide (i.e., -NH-(C=O)CH2) to the lysine of E; (e) cysteine-maleimide linkage between the maleimide in the terminal group and the cysteine ​​of E; (f) cysteine-maleimide linkage between the terminal group and the carbohydrate of E. (i) an oxime linkage between the terminal group and a carbohydrate residue of E (e.g., a carbohydrate residue of an Fc domain, albumin, or an albumin binding domain); (ii) an oxime linkage between the terminal group and an amino acid residue of E; (iii) an azido linkage between the terminal group and E; (iv) direct acylation of the terminal group to E; or (v) a thioether linkage between the terminal group and E.

[0188] Alternatively, site-specific labeling is performed by tyrosine-specific site mutation or tyrosine tag, which is genetically fused to the C-terminus of E. Tyrosinase oxidizes the phenol structure of tyrosine to quinone, allowing cycloaddition with various bicyclo[6.1.0]nonyne (BCN) derivatives, trans-cyclooctene (TCO) derivatives, etc., to link E to D.

[0189] The present disclosure also provides a pharmaceutical composition comprising at least one of the aforementioned antiviral conjugates and a pharmaceutically acceptable carrier, diluent or excipient.

[0190] In certain embodiments, the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.

[0191] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of the aforementioned antiviral conjugate based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned antiviral conjugate. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned antiviral conjugate. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned antiviral conjugate. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned antiviral conjugate.

[0192] In certain embodiments, the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent, or excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent, or excipient.

[0193] The present disclosure also provides the use of the antiviral conjugate or pharmaceutical composition described herein in the preparation of a medicament for treating a viral infection. In some embodiments, the viral infection is caused by an influenza virus or a parainfluenza virus. In some embodiments, the viral infection is caused by influenza virus A, B, or C, or a parainfluenza virus.

[0194] The present disclosure further provides a method for treating viral infection, wherein the mammal may be a human or a non-human mammal, for therapeutic purposes, comprising administering the antiviral conjugate or pharmaceutical composition of the present disclosure to the mammal.

[0195] The present disclosure further provides a kit comprising the antiviral conjugate or pharmaceutical composition described in the present disclosure.

[0196] Explanation of terms:

[0197] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0198] When a trade name is used in this disclosure, applicant intends to include the formulations of the trade name product, the generic version of the trade name product, and the active drug portion of the trade name product.

[0199] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0200] The terms "linker," "linker unit," "linker unit," "linker" or "linker fragment" refer to a chemical structure fragment or bond that is connected to a protein or polypeptide at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

[0201] The term "amino acid" refers to organic compounds containing an amino group and a carboxyl group directly attached to a -CH- structure. The general formula is H2NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Depending on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids are classified as α, β, γ, δ, ε, etc. In the biological world, the amino acids that constitute natural proteins have specific structural characteristics, that is, their amino groups are directly connected to the α-carbon atom, i.e., α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, proline, etc. Non-natural amino acids such as citrulline. As is well known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of antibodies disclosed herein. The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0202] The term "drug loading" refers to the average number of drugs carried by each conjugate molecule in a conjugate population, and can also be expressed as the ratio of the drug amount to the protein or polypeptide. The range of drug loading can be 1-20, such as 1-15, such as 1-10 neuraminidase inhibitors (D) connected to each protein or polypeptide. In an embodiment of the present disclosure, drug loading is expressed as n, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the mean of any two values. For example, 1-10, such as 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5 mean. The average amount of drug per conjugate after the conjugation reaction can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, mAb size variant determination (CE-SDS) and HPLC characterization.

[0203] The disclosed monoclonal antibody size variant determination method (CE-SDS) can employ capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) ultraviolet detection to quantitatively determine the purity of recombinant monoclonal antibody products based on molecular weight under reducing and non-reducing conditions according to capillary electrophoresis (Chinese Pharmacopoeia 2015 edition, 0542).

[0204] In one embodiment of the present disclosure, the neuraminidase inhibitor is coupled to the N-terminal amino group and / or the ε-amino group of the lysine residue of the ligand via a linker. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0205] The loading capacity of the conjugate can be controlled by the following non-limiting methods, including:

[0206] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0207] (2) Control reaction time and temperature,

[0208] (3) Select different reaction reagents.

[0209] The term "antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0210] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

[0211] The antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, such as humanized antibodies and fully human antibodies.

[0212] The term "murine antibody" as used herein refers to antibodies produced in mice according to the knowledge and skills in the art. During production, a test subject is injected with a specific antigen and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0213] The term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the mouse hybridoma cells. Furthermore, the constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are then linked to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0214] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the antibody variable region framework of a human, i.e., different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components they carry. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk / vbase), as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in immunogenicity and a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after CDRs have been affinity matured by phage display. References further describing methods for humanizing mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the methods of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al., Nature, 332, 323-327 (1988), Verhoeyen, et al., Science, 239, 1534 (1988)].

[0215] The terms "fully human antibody", "fully human antibody" or "completely human antibody", also known as "fully human monoclonal antibody", are antibodies whose variable and constant regions are both human, eliminating immunogenicity and toxic side effects. The development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies. The present disclosure is a fully human monoclonal antibody. The relevant technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology, etc.

[0216] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) a combination of two or more isolated CDRs, optionally connected by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, so that they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0217] Fab is an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), in which approximately half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

[0218] F(ab')2 is an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin, and comprises two Fab regions linked at the hinge position.

[0219] Fab' is an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F(ab')2.

[0220] Furthermore, the Fab' fragment of the antibody can be produced by inserting a DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.

[0221] The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example, variants using 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0222] The term "CDR" refers to one of the six hypervariable regions that mainly contribute to antigen binding in the variable domains of an antibody. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR is only applied to the CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and the CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), among others. For example, for the classical format, following the Kabat convention, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the Chothia convention, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).By combining the CDR definitions of Kabat and Chothia, CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Following the IMGT rules, the CDR amino acid residues in VH are numbered approximately as 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately as 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align.

[0223] The term "Fc domain monomer" or "Fc monomer" refers to a polypeptide chain comprising at least a hinge domain and second and third antibody constant domains (C H 2 and C H 3) or a functional fragment thereof (e.g., capable of (i) dimerizing with another Fc domain monomer to form an Fc domain, and (ii) binding to an Fc receptor, or a fragment thereof). In some embodiments, an Fc domain monomer comprises, from N-terminus to C-terminus, a hinge domain, a C H 2 and a C H3. The Fc domain monomer can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA or IgD (e.g., IgG). In addition, the Fc domain monomer can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3 or IgG4) (e.g., IgG1). The Fc domain monomer in the conjugate as described herein can contain one or more changes relative to the wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions or deletions) that change the interaction between the Fc domain and the Fc receptor. Examples of suitable changes are known in the art. In certain embodiments, a human Fc domain monomer (e.g., an IgG heavy chain, such as IgG1) comprises a region extending from any one of Asn201, Asn208, Glu216, Asp221, Lys222 or Cys226 to the heavy chain carboxyl terminus at Lys447. The C-terminal Lys447 of the Fc region may or may not be present and does not affect the structure or stability of the Fc region. Unless otherwise specified herein, the numbering of amino acid residues in the IgG or Fc domain monomer is according to the EU numbering system for antibodies, which is also called the Kabat EU index, as described in, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0224] The term "antibody framework" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0225] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-continuous amino acids in a unique spatial conformation (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

[0226] The term "albumin" refers to a polypeptide comprising amino acids corresponding to naturally occurring albumin (e.g., human serum albumin) or variants thereof (e.g., engineered variants of naturally occurring albumin). Variants of albumin include polymorphisms, fragments such as domains and subdomains, and fusion proteins (e.g., albumin with a C-terminal or N-terminal fusion such as a polypeptide linker). Albumin may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. In some embodiments, non-naturally occurring amino acid residues (e.g., side chains of non-naturally occurring amino acid residues) may serve as points of attachment for compounds of the disclosure (e.g., neuraminidase inhibitor monomers or dimers, including via a linker).

[0227] The term "albumin binding domain" encompasses polypeptides that have albumin binding activity, as well as antibodies or fragments thereof that bind to albumin. An "albumin binding domain" has an affinity for and ability to bind to albumin (e.g., human serum albumin). Albumin binding domains can have different origins, such as synthetic or derived from humans, mice, or rats. Albumin binding domains can be linear or cyclic, and include any albumin binding domain known to those skilled in the art. Exemplary albumin binding domains are described in US 2005 / 0287153, which is incorporated herein in its entirety.

[0228] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example: approximately less than 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0229] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, for example, double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0230] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, a vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, a vector is a viral vector, in which another DNA segment can be connected to a viral genome. Vectors disclosed herein can autonomously replicate in the host cell into which they have been introduced (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after introducing the host cell, thereby replicating (e.g., non-additional mammalian vectors) with the host genome.

[0231] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as those described in Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the present invention utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website (http: / / imgt.cines.fr) by comparing the IMGT Human Antibody Variable Region Germline Gene Database with MOE software, or from the Journal of Immunoglobulins, 2001 ISBN 012441351.

[0232] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plants, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) and NSO cells.

[0233] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended existing technology, mammalian expression systems will lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product should be immediately frozen, such as at -70°C, or freeze-dried.

[0234] Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence, after aligning the amino acid sequences and, if necessary, introducing gaps to achieve maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining amino acid sequence identity percentage, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0235] The term "peptide" refers to a compound fragment between amino acids and proteins, which is composed of two or more amino acid molecules connected by peptide bonds. It is a structural and functional fragment of protein. Hormones, enzymes, etc. are essentially peptides.

[0236] The term "sugar" refers to biological macromolecules composed of three elements: C, H, and O, which can be divided into monosaccharides, disaccharides, and polysaccharides.

[0237] The term "fluorescent probe" refers to a class of fluorescent molecules that have characteristic fluorescence in the ultraviolet-visible-near-infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can sensitively change with the properties of the environment, such as polarity, refractive index, viscosity, etc. They interact non-covalently with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescent properties, and can be used to study the properties and behavior of macromolecular substances.

[0238] "Alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched groups of 1 to 20 carbon atoms. Alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl and various branched isomers thereof. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino (this needs to be adjusted according to the claims!).

[0239] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyls include spirocyclic, paracyclic, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino (this needs to be adjusted according to the claims!).

[0240] The term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 7 ring atoms. Non-limiting examples of "heterocycloalkyl" include:

[0241] etc.

[0242] The heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

[0243] wait.

[0244] Heterocycloalkyl may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 6-membered cycloalkyl or 3- to 6-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino (this needs to be adjusted according to the claims!).

[0245] The term "alkoxy" refers to -O-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, 3- to 7-membered cycloalkyl or 3- to 7-membered heterocycloalkyl, wherein the alkyl, alkoxy, cycloalkyl or heterocycloalkyl is optionally substituted by halogen, hydroxy, nitro, cyano or amino (this needs to be adjusted according to the claims!).

[0246] Similarly, "cycloalkoxy" and "heterocycloalkoxy" are the same as the above-mentioned "alkoxy".

[0247] The term "alkylthio" refers to -S-(alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methylthio, ethylthio, propylthio, butylthio. Alkylthio may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups, independently selected from C 1-6 Alkoxy, 3 to 6 membered cycloalkyl, 3 to 6 membered heterocycloalkyl, 3 to 6 membered cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 1-6 Alkylthio, 3- to 6-membered cycloalkylthio, 3- to 6-membered heterocycloalkylthio, wherein the alkoxy, cycloalkyl, heterocycloalkyl, cycloalkyloxy, heterocyclooxy, alkylthio, cycloalkylthio, heterocycloalkylthio are optionally substituted with halogen, hydroxy, cyano or amino (this needs to be adjusted according to the claims!).

[0248] Similarly, "cycloalkylthio" and "heterocycloalkylthio" are the same as defined above for "alkylthio".

[0249] A "monovalent group" is a compound formed by formally eliminating a monovalent atom or group. A "subunit" is a compound formed by formally eliminating two monovalent or one divalent atom or group.

[0250] The term "alkylene" refers to the portion remaining after removing two hydrogen atoms from an alkane molecule, including straight and branched subgroups of 1 to 20 carbon atoms. Non-limiting examples of alkylene groups containing 1 to 6 carbon atoms include methylene (-CH2-), ethylene (such as -CH2CH2- or -CH(CH3)-). Unless otherwise specified, alkylene groups may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available attachment point, preferably one or more of the following groups, independently selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl or C 1-6 Alkoxy (this needs to be adjusted according to the claims!).

[0251] Similarly, the definitions of "alkyleneoxy", "alkenylene", "alkenyleneoxy", "cycloalkylene" and "heterocycloalkylene" are the same as "alkylene".

[0252] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0253] Aryl may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, 3- to 6-membered cycloalkoxy, 3- to 6-membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, 3 to 6 membered cycloalkoxy, 3 to 6 membered heterocycloalkoxy, 3 to 8 membered cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl are optionally substituted by one or more selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl or C 1-6 Alkoxy (this needs to be adjusted according to the claims!).

[0254] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 6 to 12-membered, more preferably 5-membered or 6-membered. For example, non-limiting examples include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.

[0255] The heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0256] Heteroaryl may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl or C 1-6 Alkoxy (this needs to be adjusted according to the claims!).

[0257] The term "spirocyclic" refers to a compound in which two rings share one atom. Non-limiting examples of spiroalkyl groups include:

[0258] The term "cycloalkyl" refers to a compound in which two or more rings are joined by sharing two adjacent atoms. Non-limiting examples of cycloalkyl groups include:

[0259] The term "bridged ring" refers to a structure formed by two or more cyclic structures sharing two non-adjacent ring atoms. Depending on the number of constituent rings, bridged cycloalkyl groups can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0260] The term "heterocycle" refers to a ring having atoms other than carbon atoms, and includes heterocycloalkyl and heteroaryl rings.

[0261] The term "hydroxy" refers to an -OH group.

[0262] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0263] The term "cyano" refers to -CN.

[0264] The term "amino" refers to -NH2.

[0265] The term "nitro" refers to -NO2.

[0266] The term "oxo" refers to a =0 substituent.

[0267] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently replaced by a corresponding number of substituents. When the substituent is keto or oxo (i.e., =O), then two (2) hydrogen atoms on the atom are replaced.

[0268] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocycloalkyl group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and that the description includes instances where the heterocycloalkyl group is substituted with an alkyl group and instances where the heterocycloalkyl group is not substituted with an alkyl group.

[0269] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0270] The term "drug carrier" as used in the context of the present disclosure refers to a system that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, preferably micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules while having good membrane permeability, making them excellent drug carriers.

[0271] The term "excipient" refers to any additive in a pharmaceutical preparation other than the main drug, also known as an adjuvant. Examples include binders, fillers, disintegrants, and lubricants in tablets; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0272] The term "diluent," also known as filler, primarily increases the weight and volume of a tablet. The addition of a diluent not only maintains a certain volume, but also reduces dosage variations of the primary ingredient and improves the drug's compressibility. When the tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production.

[0273] The compounds disclosed herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and may be defined in terms of absolute stereochemistry as (R)- or (S)-, or for amino acids, (D)- or (L)-, other stereoisomeric forms. The disclosure includes all possible isomers and their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or may be prepared using conventional methods such as chromatography and fractional crystallization. Conventional methods for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemates (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise indicated, it is intended that the compounds include both E and Z geometric isomers. Furthermore, all tautomeric forms are also meant to be included.

[0274] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Two configurations. In the chemical structure of the compound disclosed in the present invention, the bond The configuration is not specified, that is, it can be Z configuration or E configuration, or contain both configurations.

[0275] "Stereoisomers" refer to compounds composed of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. Various stereoisomers and mixtures thereof are contemplated in this disclosure, and include "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0276] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. Included in this disclosure are tautomers of any of the described compounds.

[0277] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N.15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0278] Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have at least 1000 times the abundance of deuterium greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium incorporation). In the example, the compound has a natural abundance greater than deuterium that can be at least 1000 times the abundance of deuterium, at least 2000 times the abundance of deuterium, at least 3000 times the abundance of deuterium, at least 4000 times the abundance of deuterium, at least 5000 times the abundance of deuterium, at least 6000 times the abundance of deuterium or more abundant deuterium. The disclosure also includes various deuterated forms of formula (I) compounds. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of formula (I) compounds with reference to relevant literature. Commercially available deuterated starting materials may be used in the preparation of deuterated forms of the compounds of formula (I), or they may be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0279] FIG1 shows the measured cellular levels of cytotoxicity and anti-influenza virus function of the conjugate molecules disclosed herein.

[0280] Figure 2 shows the PK curves of different test products after a single intravenous injection in cynomolgus monkeys. DETAILED DESCRIPTION

[0281] The preparation of the compounds and pharmaceutically acceptable salts disclosed herein is further described below with reference to examples, but these examples are not intended to limit the scope of the disclosure.

[0282] Experimental methods in the examples of this disclosure that do not specify specific conditions are generally based on conventional conditions or the conditions recommended by the raw material or product manufacturers. Reagents without specific sources are conventional reagents purchased from the market.

[0283] NMR shift (δ) was 10 -6 The NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, and the solvent used was deuterated dimethyl sulfoxide (DMSO-d). 6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and tetramethylsilane (TMS) as the internal standard.

[0284] MS was measured using a Shimadzu 2010 Mass Spectrometer or an Agilent 6110A MSD mass spectrometer.

[0285] HPLC determination was performed using a Shimadzu LC-20A systems, Shimadzu LC-2010HT series, or Agilent 1200LC high-pressure liquid chromatograph (Ultimate XB-C18 3.0*150 mm column or Xtimate C18 2.1*30 mm column).

[0286] Chiral HPLC analysis was performed using Chiralpak IC-3 100×4.6mm ID, 3um, Chiralpak AD-3 150×4.6mm ID, 3um, Chiralpak AD-3 50×4.6mm ID, 3um, Chiralpak AS-3 150×4.6mm ID, 3um, Chiralpak AS-3 100×4.6mm ID, 3μm, ChiralCel OD-3 150×4.6mm ID, 3um, Chiralcel OD-3 100×4.6mm ID, 3μm, ChiralCel OJ-H 150×4.6mm ID, 5um, Chiralcel OJ-3 150×4.6mm ID, 3um columns;

[0287] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm~0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm~0.5mm.

[0288] Column chromatography generally uses Yantai Huanghai silica gel 100-200 mesh, 200-300 mesh or 300-400 mesh silica gel as the carrier.

[0289] The chiral preparative column used was DAICEL CHIRALPAK IC (250 mm*30 mm, 10 um) or Phenomenex-Amylose-1 (250 mm*30 mm, 5 um).

[0290] The CombiFlash rapid preparation instrument used was Combiflash Rf150 (TELEDYNE ISCO).

[0291] Average kinase inhibition rate and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG, Germany).

[0292] The known starting materials disclosed herein can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals, and other companies.

[0293] Unless otherwise specified in the examples, all reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0294] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0295] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0296] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0297] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0298] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0299] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0300] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0301] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the column chromatography eluent system used for purifying the compound, and the developing solvent system for thin layer chromatography included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, C: petroleum ether / ethyl acetate system, and D: petroleum ether / ethyl acetate / methanol. The volume ratio of the solvent was adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0302] The abbreviations used in the following experiments have the following meanings:

[0303] EtOAc: ethyl acetate; DCM: dichloromethane; DIPEA: N,N-diisopropylethylamine; PPTS: pyridinium p-toluenesulfonate; Boc: tert-butyloxycarbonyl, MeOH: methanol.

[0304] PEG4-azido NHS ester:

[0305] Azido-PEG3-Maleimide:

[0306] N3-PEG3-Oxa:

[0307] Example 1 Synthesis of Intermediate Int-A

[0308] Step 1:

[0309] Compound (1S,2R)-1-((2R,3R,4S)-3-acetamido-4-azido-6-(methoxycarbonyl)-3,4-dihydro-2H-pyran-2-yl)propane-1,2,3-triacetate triester Int-A-1 (8.5 g, 18.6 mmol) was dissolved in a mixture of ethanol (90 mL) and water (15 mL). Ammonium chloride (3.0 g, 56 mmol) and zinc powder (3.9 g, 60 mmol) were added sequentially. The reaction was stirred at 30°C for 30 minutes. After completion of the reaction, the reaction mixture was cooled to 0°C and filtered. The filtrate was concentrated, and ethyl acetate (100 mL) was added. The mixture was then filtered and concentrated to afford compound Int-A-2 (8.0 g, 100% yield).

[0310] 1 H NMR (400MHz, CDCl3): δ6.14(s,1H),5.49(s,1H),5.29(s,1H),4.61(d,1H),4.50–4.15(m,2H),3.78-3.71(m,4H),2.10–2.04(m,12H).

[0311] Step 2:

[0312] Int-A-2 (8.0 g, 18.6 mmol) was dissolved in dry tetrahydrofuran (80 mL), and N,N'-di-BOC-1H-1-guanidinopyrazole (6.3 g, 20.5 mmol) and triethylamine (4 mL, 27.9 mmol) were added sequentially. The reaction was stirred at room temperature overnight. After completion, the reaction mixture was concentrated and ethyl acetate (200 mL) was added. The mixture was then washed sequentially with water (100 mL) and saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound Int-A-4 (11 g, 87.9% yield).

[0313] 1 H NMR (400MHz, CDCl3): δ11.40(s,1H),8.51(d,1H),6.75(br,1H),5.86(s,1H),5.46(d,1H),5.31–5.24(m,1H),5.12(t, 1H),4.64(dd,1H),4.30–4.12(m,3H),3.77(s,3H),2.10(s,3H),2.05(s,3H),2.03(s,3H),1.84(s,3H),1.46(d,18H).

[0314] Step 3:

[0315] Dissolve compound Int-A-4 (12.3 g, 18.3 mmol) in anhydrous methanol (240 mL) and add sodium methoxide solution (0.5 M in MeOH, 6.5 mL, 3.25 mmol). Stir the reaction at room temperature for 30 minutes. After completion, neutralize the reaction with IRN-77 resin to a neutral pH. The neutralized reaction solution is filtered and concentrated to afford compound Int-A-5 (9.0 g, crude product), which is used directly in the next step.

[0316] 1 H NMR (400MHz, CDCl3): δ11.37(s,1H),8.59(d,1H),8.23(d,1H),5.80(s,1H),5.36–5.18(m, 2H),4.21(d,1H),4.11–3.82(m,5H),3.79(s,3H),3.61(d,1H),2.02(s,3H),1.50(d,18H).

[0317] Step 4:

[0318] Compound Int-A-5 (9.0 g, crude product) was dissolved in acetone (180 mL), and 2,2-dimethoxypropane (24 mL, 196 mmol) and p-toluenesulfonic acid monohydrate (740 mg, 3.92 mmol) were added sequentially. The reaction was stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound Int-A-6 (8.5 g, 79.2% yield over two steps).

[0319] 1 H NMR (400MHz, CDCl3): δ11.36(s,1H),8.65(d,1H),8.02(d,1H),5.80(d,1H),5.27(s,1H),5.16(t,1H),4.43–4.36(m,1H ),4.21–4.07(m,2H),4.05–3.93(m,2H),3.79(s,3H),3.50(d,1H),2.01(s,3H),1.50(d,18H),1.43(s,3H),1.37(s,3H).

[0320] Step 5:

[0321] Compound Int-A-6 (8.2 g, 14.0 mmol) was dissolved in dry dichloromethane (160 mL). 4-Dimethylaminopyridine (13.7 g, 112 mmol) was added under an ice bath, followed by the addition of 4-nitrophenyl chloroformate (16.8 g, 84 mmol) in batches. The reaction was stirred at room temperature overnight. After completion, the reaction solution was concentrated. The crude product was diluted with ethyl acetate to precipitate a solid, which was then filtered. The filtrate was concentrated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to afford compound Int-A-7 (5.2 g, 49.4% yield).

[0322] 1 H NMR (400MHz, CDCl3): δ11.35(s,1H),8.56(d,1H),8.35(d,2H),7.54(d,2H),6.60(d,1H),5.90(d,1H),5.26(d,1H),5.23–5.16(m ,1H),4.49–4.37(m,2H),4.27–4.20(m,2H),4.18–4.12(m,1H),3.81(s,3H),1.92(s,3H),1.49(s,18H),1.42(s,3H),1.39(s,3H).

[0323] Step 6:

[0324] To a solution of compound Int-A-8 (5.0 g, 24.4 mmol) in tetrahydrofuran was added a 2.5 M solution of lithium aluminum tetrahydride in tetrahydrofuran (29.2 mL, 73.0 mmol) at 0°C. After the addition was complete, the mixture was heated to 80°C and stirred overnight. After the reaction was complete, the cooled reaction solution was slowly poured into a solution of sodium sulfate decahydrate (10 g) in tetrahydrofuran (30 mL) and stirred at room temperature for one hour. The solid was removed by filtration, and the mother liquor was dried to give compound Int-A-9 (2.90 g, crude product). This product was used directly in the next step without further purification.

[0325] MS m / z(ESI):120.2[M+1] + .

[0326] Step 7:

[0327] To a solution of compound Int-A-9 (2.90 g, crude) in dichloromethane (50 mL) was added a solution of di-tert-butyl dicarbonate (6.67 g, 30.5 mmol) in dichloromethane (20 mL) at 0°C, and the mixture was stirred overnight at room temperature. The reaction mixture was concentrated and then purified by silica gel column chromatography (dichloromethane / ethyl acetate = 10 / 1 to 2 / 1) to afford compound Int-A-10 (5.27 g, two-step yield: 98.5%).

[0328] 1 H NMR (400MHz, CDCl3): δ3.77–3.68(m,2H),3.60-3.55(m,4H),3.42(s,2H),2.91(s,3H),1.46(s,9H).

[0329] Step 8:

[0330] To a solution of compound Int-A-10 (4.17 g, 19.0 mmol) in dichloromethane (60 mL) at 0°C was added triphenylphosphine (1.79 g, 28.5 mmol). Stirring was continued for 15 minutes, followed by the addition of carbon tetrabromide (9.45 g, 28.5 mmol) in portions. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was directly dried and purified by silica gel column chromatography (dichloromethane / ethyl acetate = 10 / 1 to 2 / 1) to afford compound Int-A-11 (4.14 g, 77.2% yield).

[0331] 1 H NMR (400MHz, CDCl3): δ3.77(t,2H),3.61(s,2H),3.50-3.41(m,4H),2.93(s,3H),1.46(s,9H).

[0332] Step 9:

[0333] To a solution of compound Int-A-11 (1.16 g, 4.11 mmol) in N,N-dimethylformamide (20 mL) were added compound Int-A-12 (1.00 g, 4.32 mmol) and potassium carbonate (0.896 g, 6.48 mmol), followed by heating and stirring at 50°C overnight. The reaction mixture was concentrated to remove N,N-dimethylformamide, and water (10 mL) was added. The mixture was extracted with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and then spin-dried. The mixture was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-A-13 (0.860 g, 49.9% yield).

[0334] 1 H NMR (400MHz, CDCl3): δ5.24(s,1H),4.21(s,2H),3.75-3.64(m,13H),3.61(t,2H) ),3.52(t,2H),3.35-3.26(m,2H),2.95-2.82(m,4H),2.45(s,1H),1.44(s,9H).

[0335] Step 10:

[0336] To a solution of compound Int-A-13 (0.76 g, 1.82 mmol) in dichloromethane (5 mL) was added a 4 M solution of HCl in dioxane (10 mL) at 0°C. The mixture was then stirred at room temperature for 2 hours and concentrated to afford compound Int-A-14 (0.72 g, crude product). This was used directly in the next step without further purification.

[0337] 1 H NMR (400MHz, DMSO-d6): δ8.80(br,2H),8.02(br,3H),4.15(s,2H),3.78–3.73(m,2H) ,3.72–3.67(m,2H),3.64–3.52(m,14H),3.45(t,1H),3.25-3.15(m,4H),3.02(q,2H).

[0338] Step 11:

[0339] To a solution of compound Int-A-14 (0.56 g, 1.42 mmol) in N,N-dimethylformamide (10 mL) were added compound Int-A-11 (1.98 g, 8.76 mmol) and potassium carbonate (1.21 g, 8.76 mmol), and then heated and stirred at 60°C overnight. The reaction solution was concentrated under reduced pressure to remove N,N-dimethylformamide, and water (15 mL) was added, followed by extraction with dichloromethane (15 mL x 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and spin-dried. The product was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1-10 / 1) and high performance liquid chromatography (Waters XBridge, 19 * 150 mm, 30 * 150 mm, 5 μm; mobile phase A: 0.5% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain compound Int-A-15 (587 mg, yield 36.3%).

[0340] Step 12:

[0341] To a solution of compound Int-A-15 (280 mg, 0.303 mmol) in dichloromethane (2.0 mL) was added a solution of HCl in dioxane (4 M, 5.0 mL) at 0°C. The mixture was then stirred at room temperature for 2 hours and concentrated to afford compound Int-A-16 (0.245 g, crude product). This was used directly in the next step without further purification.

[0342] MS m / z(ESI):622.6[M+1] + .

[0343] Step 13:

[0344] Compound Int-A-16 (100 mg crude product, 0.124 mmol) was dissolved in dry N,N-dimethylformamide (6 mL). N,N-diisopropylethylamine (370 mg, 2.9 mmol) was added under ice-cooling, followed by compound Int-A-7 (380 mg, 0.51 mmol). The reaction was stirred at room temperature overnight. After completion, ethyl acetate (15 mL) was added, followed by washing with water (10 mL) and saturated brine (15 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate:methanol = 8:1) to obtain compound Int-A-17 (140 mg, 45.9% yield).

[0345] 1H NMR (400MHz, CDCl3): δ11.39(s,3H),8.41(d,3H),6.34–6.10(m,3H),5.8 9(s,3H),5.31–5.20(m,6H),4.43-4.02(m,9H),4.17–4.00(m,10H),3.79( s,9H),3.70–3.47(m,34H),3.28-3.21(m,3H),3.00–2.93(m,9H),2.78-2 .62(m,10H),1.89(s,9H),1.48(d,54H),1.43(s,3H),1.38–1.34(m,15H).

[0346] Step 14:

[0347] Compound Int-A-17 (140 mg, 0.057 mmol) was dissolved in methanol (10 mL). A 10 mL aqueous solution of lithium hydroxide monohydrate (22 mg, 0.52 mmol) was added under ice. The reaction was stirred at room temperature for 16 hours. After completion, IRN 77 resin was added to neutralize the mixture to a neutral pH. The neutralized reaction solution was filtered and concentrated to afford compound Int-A-18 (110 mg, 80% yield).

[0348] MS m / z(ESI):806.6([M+3] / 3) + .

[0349] Step 15:

[0350] Compound Int-A-18 (110 mg, 0.045 mmol) was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (6 mL) was added. The reaction was stirred at room temperature for 2 hours. After completion, the mixture was spin-dried and then water (3 mL) was added and stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by HPLC (Waters XBridge, 19*150 mm, 30*150 mm, 5 μm; mobile phase A: 0.5% TFA in water, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain the title product Int-A (30.1 mg, 31% yield).

[0351] MS m / z(ESI):848.7([M+2] / 2) + ,566.0([M+3] / 3) + ;

[0352] 1H NMR (400MHz, CD3OD): δ5.83(s,3H),4.93(d,3H),4.51(d,3H),4.42-4.33(m,3H),4.24–4.14(m, 5H),4.09-3.76(m,17H),3.7-3.41(m,37H),3.22-3.13(m,4H),2.94-2.81(m,9H),1.87(s,9H).

[0353] Example 2 Synthesis of Intermediate Int-B

[0354] Step 1:

[0355] To a solution of compound Int-B-1 (3.0 g, 13.4 mmol) in N,N-dimethylformamide (45 mL) were added compound Int-A-12 (3.1 g, 13.4 mmol) and potassium carbonate (3.93 g, 28.5 mmol). The reaction mixture was then stirred and heated at 55°C overnight. The N,N-dimethylformamide was removed by concentration under reduced pressure, and water (60 mL) was added. The mixture was extracted with dichloromethane (60 mL x 3). The combined organic phases were washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The mixture was then purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-B-2 (0.810 g, 16.1% yield).

[0356] 1 H NMR(400MHz, CDCl3)δ:5.16(br,1H),4.21(s,2H),3.71–3.59(m,14H),3.28-3.21(m,2H),2.71-2.82(m,4H),2.44(s,1H),1.44(s,9H).

[0357] Step 2:

[0358] To a solution of compound Int-B-2 (800 mg, 2.14 mmol) in dichloromethane (5.0 mL) was added a solution of HCl in dioxane (4 M, 10 mL) at 0°C. The system was stirred at room temperature for 4 hours and concentrated to afford compound Int-B-3 (0.75 g, crude product), which was used directly in the next step.

[0359] Step 3:

[0360] To a solution of compound Int-B-3 (0.677 g, 2.0 mmol) in N,N-dimethylformamide (10 mL) were added compound Int-A-11 (2.20 g, 7.8 mmol) and potassium carbonate (1.35 g, 9.8 mmol), followed by heating and stirring at 65°C overnight. The reaction mixture was concentrated under reduced pressure to remove N,N-dimethylformamide, and water (15 mL) was added. The mixture was extracted with DCM (15 mL x 3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and then purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-B-4 (970 mg, 56.6% yield).

[0361] MS m / z(ESI):439.9([M+2] / 2) + .

[0362] Step 4:

[0363] To a solution of compound Int-B-4 (406 mg, 0.462 mmol) in dichloromethane (1.0 mL) was added a solution of HCl in dioxane (4 M, 6.0 mL) at 0°C. The mixture was then stirred at room temperature for 1 hour and concentrated to afford compound Int-B-5 (355 mg, crude product), which was used directly in the next step.

[0364] MS m / z(ESI):578.7[M+1] + .

[0365] Step 5:

[0366] Compound Int-B-5 (215 mg, 0.28 mmol) was dissolved in dry N,N-dimethylformamide (6 mL). N,N-diisopropylethylamine (1 mL, 6.2 mmol) was added under ice-cooling, followed by compound Int-B-5 (940 mg, 1.25 mmol). The reaction was stirred at room temperature for 16 h. After completion of the reaction, ethyl acetate (30 mL) was added, followed by washing with water (15 mL) and brine (15 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by preparative chromatography (ethyl acetate:methanol = 8:1) to obtain compound Int-B-6 (170 mg, 25.1% yield).

[0367] 1H NMR (400MHz, CDCl3): δ11.39(s,3H),8.41(d,3H),6.37-6.12(m,3H),5.89(s,3H),5.43-5.15(m,6H),4.47-3.95(m,15H) ,3.79(s,9H),3.72–3.46(m,30H),3.38-3.12(m,3H),3.05-2.55(m,23H),1.89(s,9H),1.48(s,54H),1.39–1.33(m,18H).

[0368] Step 6:

[0369] Compound Int-B-6 (140 mg, 0.058 mmol) was dissolved in methanol (10 mL), and a solution of lithium hydroxide monohydrate (22 mg, 0.51 mmol) in water (10 mL) was added at 0°C. The reaction was stirred at room temperature for 16 hours. After completion, IRN 77 resin was added to neutralize the mixture to a neutral pH. The neutralized reaction solution was filtered and concentrated to afford compound Int-B-7 (150 mg, crude), which was directly used in the next reaction.

[0370] MS m / z(ESI):792.0([M+3] / 3) + .

[0371] Step 7:

[0372] The compound Int-B-7 (150 mg, crude product) from the previous step was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (4 mL) was added. The reaction was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was spin-dried, followed by the addition of water (4 mL) and stirring at room temperature for 1 hour. The reaction solution was concentrated and purified by high-performance liquid chromatography (Waters XBridge, 19*150 mm, 30*150 mm, 5 μm; mobile phase A: 0.5% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain the title product Int-B (61.2 mg, two-step yield 47.5%).

[0373] MS m / z(ESI):827.1([M+2] / 2) + ,551.8([M+3] / 3) + ;

[0374] 1H NMR (400MHz, CD3OD): δ5.90(s,3H),5.01(d,3H),4.54(d,3H),4.40(d,3H),4.26–4.16(m,5H) ,4.04–3.97(m,3H),3.88–3.55(m,36H),3.53–3.12(m,14H),3.03-2.77(m,10H),1.95(s,9H).

[0375] Example 3 Synthesis of Intermediate Int-C

[0376] Step 1:

[0377] To a solution of compound Int-C-1 (5.0 g, 20 mmol) in dichloromethane (100 mL) at 0°C was added triphenylphosphine (7.89 g, 30 mmol). Stirring was continued and carbon tetrabromide (9.97 g, 30 mmol) was added portionwise. The mixture was stirred at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane / ethyl acetate = 10 / 1 to 4 / 1) to afford compound Int-C-2 (4.70 g, 75.3% yield).

[0378] 1 H NMR (400MHz, CDCl3): δ5.01(br,1H),3.82(t,2H),3.66-3.62(m,4H),3.55(t,2H),3.49(t,2H),3.33-3.32(m,2H),1.45(s,9H).

[0379] Step 2:

[0380] To a solution of compound Int-C-2 (2.70 g, 8.64 mmol) in N,N-dimethylformamide (40 mL) were added compound Int-A-12 (2 g, 8.64 mmol) and potassium carbonate (2.39 g, 17.30 mmol). The reaction system was heated and stirred at 55°C overnight. The reaction solution was concentrated to remove N,N-dimethylformamide, and water (20 mL) was added. The mixture was then extracted with dichloromethane (20 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-C-3 (2 g, 50.0% yield).

[0381] MS m / z(ESI):463.3[M+H] + .

[0382] 1H NMR (400MHz, CDCl3): δ5.24(br,1H),4.21(d,2H),3.69-3.60(m,20H),3.54(t ,2H),3.49(s,1H),3.32-3.31(m,2H),2.84(t,4H),2.44(t,1H),1.44(s,9H).

[0383] Step 3:

[0384] To a solution of compound Int-C-3 (1.50 g, 3.24 mmol) in dichloromethane (5 mL) was added a 4 M hydrogen chloride / dioxane solution (20 mL) at 0°C. The reaction system was stirred at room temperature for 2 hours and concentrated to afford compound Int-C-4 (1.41 g, crude product), which was used directly in the next step.

[0385] MS m / z(ESI):363.2[M+H] + .

[0386] 1 H NMR (400MHz, DMSO-d6): δ8.89(br,2H),8.05(br,3H),4.15(d,2H),3.72(t,4H),3. 66-3.60(m,6H),3.59-3.51(m,12H),3.46(t,1H),3.21-3.10(m,4H),2.97(dd,2H).

[0387] Step 4:

[0388] To a solution of compound Int-C-4 (1.41 g, crude product from the previous step) in N,N-dimethylformamide (15 mL) were added compound Int-A-11 (4.09 g, 14.49 mmol) and potassium carbonate (2.69 g, 19.46 mmol), and the system was heated and stirred at 65°C overnight. The reaction mixture was concentrated to remove N,N-dimethylformamide, and water (15 mL) was added. The mixture was extracted with dichloromethane (15 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-C-5 (1.30 g, two-step yield: 41.5%).

[0389] MS m / z(ESI):483.9([M+2H] / 2) + .

[0390] 1H NMR (400MHz, CDCl3): δ4.20(d,2H),3.72-3.62(m,12H),3.61-3.47(m,24H),3.36(s,6H),2.90(s,9H),2.77(d,10H),2.44(s,1H),1.45(s,27H).

[0391] Step 5:

[0392] To a solution of compound Int-C-5 (400 mg, 0.41 mmol) in dichloromethane (5.0 mL) was added a 4 M hydrogen chloride / dioxane solution (10 mL) at 0°C. The reaction system was then stirred at room temperature for 1 hour and concentrated to afford compound Int-C-6 (350 mg, crude product), which was used directly in the next step.

[0393] MS m / z(ESI):666.5[M+H] + .

[0394] Step 6:

[0395] To a solution of compound Int-C-6 (350 mg, crude product from the previous step) in N,N-dimethylformamide (10 mL) were added compound Int-A-7 (933 mg, 1.24 mmol) and N,N-diisopropylethylamine (533 mg, 4.12 mmol), followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated at room temperature to remove the N,N-dimethylformamide, and the mixture was reversed to afford compound Int-C-7 (302 mg, 29.4% yield over two steps).

[0396] 1 H NMR (400MHz, CDCl3): δ11.40(s,3H),8.41(d,3H),6.21-6.06(m,4H),5.88(s,3H),5.27- 5.20(m,6H),4.41-4.35(m,6H),4.20(d,2H),4.17-3.99(m,10H),3.79(s,9H),3.69(d,4 H),3.66-3.61(m,7H),3.60-3.48(m,27H),3.28-3.21(m,4H),2.99(s,6H),2.94(s,3H), 2.75(br,10H),2.47-2.46(m,1H),1.90-1.80(m,9H),1.48(d,52H),1.40-1.33(m,18H).

[0397] Step 7:

[0398] To a solution of compound Int-C-7 (200 mg, 79.88 μmol) in methanol (11.2 mL) was added an aqueous solution of lithium hydroxide monohydrate (13.3 mg, 0.32 mmol) (11.2 mL) at 0°C, followed by stirring at 20°C for 12 hours. After completion of the reaction, the reaction solution was adjusted to pH 7 using ion exchange resin IRN77. The filtered mother liquor was concentrated to afford compound Int-C-8 (197 mg, crude product), which was used directly in the next step.

[0399] MS m / z(ESI):821.3([M+3H] / 3) + .

[0400] Step 8:

[0401] To a solution of compound Int-C-8 (197 mg, crude product from the previous step) in dichloromethane (5.0 mL) was added trifluoroacetic acid (5.0 mL) at 0°C. The mixture was then stirred at 20°C for 1 hour, concentrated, and then water (5 mL) was added. The mixture was stirred for 2 hours, concentrated under reduced pressure, and purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.5% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to give the title product, Int-C trifluoroacetate (43 mg, two-step yield: 23.3%).

[0402] MS m / z(ESI):870.9([M+2H] / 2) + .

[0403] 1 H NMR (400MHz, CD3OD): δ5.92(s,3H),5.02-5.04(m,3H),4.56(dd,3H),4.43(d,3H),4.31-4.18(m,6H),4.09-3.90( m,17H),3.74-3.68(m,22H),3.62-3.42(m,17H),3.26-3.14(m,5H),3.01(s,5H),2.98-2.92(m,4H),1.97(s,9H).

[0404] Example 4 Synthesis of Intermediate Int-D

[0405] Step 1: Synthesis of compound Int-C-3

[0406] Compound Int-A-12 (1 g, 4.32 mmol, purchased from Shaoyuan) and compound Int-C-2 (1.35 g, 4.32 mmol, prepared according to a literature method, Bioorganic and Medicinal Chemistry Letters, 2012, vol. 22, #2, p. 1151-1155) were dissolved in DMF and stirred. Potassium carbonate (1.2 g, 8.65 mmol) was added and heated to 55°C for 16 hours. The reaction solution was concentrated to dryness, and dichloromethane (20 mL) and water (10 mL) were added for separation. The aqueous phase was extracted with dichloromethane (20 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to dryness. The mixture was purified by column chromatography (DCM:MeOH = 0-10%) to obtain 1.12 g of compound Int-C-3 (yield: 55.99%).

[0407] MS m / z(ESI):463.3[M+1] + .

[0408] Step 2: Synthesis of compound Int-C-4

[0409] Compound Int-C-3 (300 mg, 0.648 mmol) was dissolved in dichloromethane (1.5 mL) and stirred in an ice-water bath. Hydrochloric acid and dioxane solution (6 mL) were added and the mixture was naturally warmed to room temperature for 2 hours. The reaction solution was concentrated to dryness to obtain 290 mg of crude product, which was used directly in the next reaction.

[0410] MS m / z(ESI):363.2[M+1] + .

[0411] Step 3: Synthesis of compound Int-D-6

[0412] Compound Int-D-5 (2.51 g, 9.53 mmol, prepared using the method of WO2019 / 195609) was dissolved in dichloromethane (30 mL) and stirred. Triphenylphosphine (3.75 g, 14.30 mmol) was added and stirred at room temperature for 15 minutes, followed by the addition of carbon tetrabromide (4.74 g, 14.30 mmol) in portions. After addition, the mixture was reacted at room temperature for 1 hour. The reaction solution was concentrated to dryness and purified by column chromatography (DCM:EA = 0-50%) to obtain 2.87 g of compound Int-D-6 (yield: 92.28%).

[0413] MS m / z(ESI):348.1[M+23] + .

[0414] Step 4: Synthesis of compound Int-D-7

[0415] Compound Int-C-4 (917 mg, 2.11 mmol) and compound Int-D-6 (2.4 g, 7.37 mmol) were dissolved in DMF (10 mL) and stirred. Potassium carbonate (2.91 g, 21.06 mmol) was added and heated to 65°C for 16 hours. The reaction solution was concentrated to dryness and then added with dichloromethane (20 mL) and water (10 mL) for separation. The aqueous phase was extracted with dichloromethane (20 mL x 2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and purified by column chromatography (DCM:MeOH = 0-10%) to obtain 1.47 g of compound Int-D-7 (yield: 63.55%).

[0416] MS m / z(ESI):550.0[M / 2+1] + .

[0417] Step 5: Synthesis of compound Int-D-8

[0418] Compound Int-D-7 (500 mg, 0.455 mmol) was dissolved in dichloromethane (5 mL) and stirred in an ice-water bath. Dioxane hydrochloride solution (12.52 mL) was added and the mixture was allowed to warm to room temperature for 2 hours. The reaction mixture was concentrated to dryness to yield 481.17 mg of crude product, which was used directly in the next reaction.

[0419] MS m / z(ESI):798.3[M+1] + .

[0420] Step 6: Synthesis of compound Int-D-9

[0421] Compound Int-D-8 (446.27 mg, 0.456 mmol) was dissolved in DMF (5 mL) and stirred. DIPEA (589.50 mg, 4.56 mmol) was added, and the mixture was stirred at room temperature for 5 minutes. Compound Int-A-7 (1.03 g, 1.37 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours. The mixture was then purified by column chromatography (ammonium bicarbonate:methanol = 70-95%) to obtain 590.9 mg of compound Int-D-9 (yield: 49.15%).

[0422] MS m / z(ESI):1318.7[M / 2+1] + .

[0423] Step 7: Synthesis of compound Int-D-10

[0424] Compound Int-D-9 (200 mg, 75.87 μmol) was dissolved in methanol (15 mL) and stirred. Water (15 mL) was added, followed by lithium hydroxide monohydrate (31.84 mg, 758.75 μmol). The mixture was allowed to react at room temperature for 16 hours. Ion exchange resin IRN77 was added to the reaction mixture, adjusting the pH to 7. The reaction mixture was then filtered, the filter cake was rinsed with methanol, and the filtrate was concentrated to dryness to yield 0.197 g of crude product, which was used directly in the next step.

[0425] MS m / z(ESI):1297.6[M / 2+1] + .

[0426] Step 8: Synthesis of compound Int-D

[0427] Compound Int-D-10 (0.197 g, 75.95 μmol) was dissolved in DCM (6 mL) and stirred at room temperature. TFA (8.88 g, 77.85 mmol) was added and stirred at room temperature for 5 hours. The reaction solution was concentrated to dryness. Water (6 mL) was added and stirred at room temperature for 3.5 hours. The mixture was then purified by column chromatography (TFA:acetonitrile = 70-85%) to obtain 117.7 mg of compound Int-D (yield: 63.43%).

[0428] MS m / z(ESI):937.3[M / 2+1] + , 625.3[M / 3+1] + , 469.1[M / 4+1] +

[0429] 1 H NMR (400MHz, CDCl3): δ5.89-5.80(m,3H),5.05-4.95(m,5H),4.58-4.49(m,3H),4.46-4.38(m,3H),4.29-4.13(m,5H), 4.09-3.96(m,3H),3.96-3.75(m,15H),3.75-3.44(m,56H),3.22-3.10(m,3H),3.02-2.89(m,10H),1.98-1.91(m,9H).

[0430] Example 5 Synthesis of Intermediate Int-E

[0431] Step 1:

[0432] To a solution of compound Int-A-12 (1.50 g, 6.49 mmol) in N,N-dimethylformamide (25 mL) were added compound Int-E-1 (3.82 g, 14.25 mmol) and potassium carbonate (2.68 g, 19.39 mmol), and the system was heated and stirred at 55°C for 12 hours. The reaction solution was concentrated to remove N,N-dimethylformamide, and water (20 mL) was added. The mixture was extracted with dichloromethane (20 mL × 3). The combined organic phases were washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 30 / 1 to 10 / 1) to afford compound Int-E-2 (1.95 g, 49.6% yield).

[0433] MS m / z(ESI):606.4[M+H] + .

[0434] 1 H NMR (400MHz, CDCl3): δ4.21(d,2H),3.74–3.58(m,14H),3.58-3.46(m,10H),3.30(d,4H),2.78-2.75(m,6H),2.44(t,1H),1.44(s,18H).

[0435] Step 2:

[0436] To a solution of compound Int-E-2 (1.85 g, 3.05 mmol) in dichloromethane (2.0 mL) was added a hydrogen chloride / dioxane solution (4 M, 10 mL) at 0°C. The mixture was then stirred at 0°C for 1 hour and concentrated to afford compound Int-E-3 (1.51 g, crude product), which was used directly in the next step.

[0437] Step 3:

[0438] To a solution of compound Int-E-3 (1.51 g, crude product from the previous step) in N,N-dimethylformamide (20 mL) were added compound Int-A-11 (4.70 g, 16.66 mmol) and potassium carbonate (2.93 g, 21.20 mmol), and the system was heated and stirred at 65°C overnight. The reaction solution was concentrated to remove the solvent, and water (15 mL) was added. The mixture was extracted with dichloromethane (15 mL x 3). The organic phase was washed with brine (20 mL), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-E-4 (1.32 g, two-step yield: 35.8%).

[0439] MS m / z(ESI):606.0[M+2H] / 2 + .

[0440] 1 H NMR (400MHz, CDCl3): δ4.20(d,2H),3.71-3.59(m,16H),3.51-3.49(m,29H),3.37(s,8H),2.90(s,12H),2.76(s,12H),1.45(s,36H).

[0441] Step 4:

[0442] To a solution of Int-E-4 (250 mg, 0.21 mmol) in dichloromethane (3.0 mL) was added hydrogen chloride / dioxane solution (4 M, 6 mL) at 0°C, followed by stirring at room temperature for 2 hours and concentration to give compound Int-E-5 (220 mg, crude product), which was directly used in the next step.

[0443] Step 5:

[0444] To a solution of Int-E-5 (220 mg, crude product from the previous step) in N,N-dimethylformamide (10 mL) were added compound Int-A-7 (619 mg, 0.82 mmol) and N,N-diisopropylethylamine (266 mg, 2.06 mmol), followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated at room temperature to remove the N,N-dimethylformamide, and the reaction mixture was reversed to afford compound Int-E-6 (270 mg, 40.1% yield over two steps).

[0445] MS m / z(ESI):816.0[M+4H] / 4 + .

[0446] 1 H NMR (400MHz, CDCl3): δ11.39(s,4H),8.41(d,4H),6.23-6.11(m,5H),5.89(s,4H),5.27 -5.10(m,8H),4.38-4.36(m,8H),4.20(d,2H),4.17-3.96(m,12H),3.79(s,12H),3.72-3 .61(m,12H),3.59-3.53(m,14H),3.49-3.47(m,20H),3.23(s,4H),2.99(s,6H),2.94(s ,4H),2.76-2.73(m,16H),2.47(t,1H),1.89(s,12H),1.48(d,72H),1.40-1.32(m,23H).

[0447] Step 6:

[0448] To a solution of Int-E-6 (200 mg, 61.34 μmol) in methanol (12.3 mL) was added an aqueous solution of lithium hydroxide monohydrate (26 mg, 0.62 mmol) (12.3 mL) at 0°C, followed by stirring at 20°C for 12 hours. After completion of the reaction, the reaction solution was adjusted to pH 7 using ion exchange resin IRN77. The filtered mother liquor was concentrated to afford compound Int-E-7 (196 mg, crude product), which was used directly in the next step.

[0449] MS m / z(ESI):802.0[M+4H] / 4 + .

[0450] Step 7:

[0451] To a solution of compound Int-E-7 (196 mg, crude product from the previous step) in dichloromethane (5 mL) was added trifluoroacetic acid (10 mL) at 0°C. The mixture was then stirred at 20°C for 2 hours, concentrated, and then water (10 mL) was added and stirred for 2 hours. The mixture was concentrated under reduced pressure and analyzed by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.5% aqueous TFA, mobile phase B: acetonitrile, gradient: phase B 5%-95%) to afford the title product, Int-E trifluoroacetate (50 mg, two-step yield: 26.9%).

[0452] MS m / z(ESI):1121.8[M+2H] / 2 + .

[0453] 1 H NMR(400MHz,D2O): δ5.99(s,4H),4.86(t,4H),4.38(t,4H),4.26(d,4H),4.15(s,2H),4.0 5-3.92(m,8H),3.69-3.42(m,53H),3.18-2.94(m,4H),2.79-2.54(m,28H),1.75(s,12H).

[0454] Example 6 Synthesis of Intermediate Int-F

[0455] Step 1:

[0456] To a solution of compound Int-F-1 (2.50 g, 8.52 mmol) in dichloromethane (40 mL) at 0°C was added triphenylphosphine (3.35 g, 12.77 mmol). Stirring was continued for 15 minutes, followed by the addition of carbon tetrabromide (4.23 g, 12.76 mmol) in portions. The mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to afford the product Int-F-2 (2.10 g, 69.2% yield).

[0457] 1 H NMR (400MHz, CDCl3): δ5.05(br,1H),3.82(t,2H),3.71-3.61(m,8H),3.55(t,2H),3.48(t,2H),3.36-3.27(m,2H),1.45(s,9H).

[0458] Step 2:

[0459] To a solution of compound Int-F-2 (2.00 g, 5.61 mmol) in N,N-dimethylformamide (20 mL) were added compound Int-A-12 (1.29 g, 5.58 mmol) and potassium carbonate (1.56 g, 11.29 mmol), followed by heating and stirring at 55°C for 6 hours. The reaction mixture was concentrated to remove N,N-dimethylformamide, and water (10 mL) was added. The mixture was extracted with dichloromethane (10 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 30 / 1 to 10 / 1) to afford compound Int-F-3 (1.38 g, 48.6% yield).

[0460] 1 H NMR (400MHz, CDCl3): δ5.26(s,1H),4.21(d,2H),3.73-3.57(m,23H),3.54(t,2H),3.31(d,2H),2.85(t,4H),2.44(s,1H),1.44(s,9H).

[0461] Step 3:

[0462] To a solution of compound Int-F-3 (1.28 g, 2.53 mmol) in dichloromethane (4.0 mL) was added a 4M hydrogen chloride / dioxane solution (10 mL) at 0°C. The mixture was then stirred at 20°C for 2 hours and concentrated to afford compound Int-F-4 hydrochloride (1.21 g, crude product), which was used directly in the next reaction.

[0463] 1H NMR (400MHz, DMSO): δ8.74(br,2H),7.93(br,3H),4.15(d,2H),3.70(t,4H),3.55(d,24H),3.46(t,1H),3.15(dd,4H),2.97(dd,2H).

[0464] Step 4:

[0465] To a solution of Int-F-4 hydrochloride (1.21 g, crude product from the previous step) in N,N-dimethylformamide (15 mL) were added Int-A-11 (2.81 g, 9.96 mmol) and potassium carbonate (2.43 g, 17.58 mmol), followed by heating and stirring at 65°C overnight. The N,N-dimethylformamide was removed by concentration, and water (15 mL) was added. The mixture was extracted with dichloromethane (15 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford Int-F-5 (1.06 g, 41.5% yield).

[0466] 1 H NMR (400MHz, CDCl3): δ4.21(d,2H),3.72-3.58(m,24H),3.53-3.37(m,22H),2.90(s,9H),2.82-2.73(m,10H),2.44(s,1H),1.45(s,27H).

[0467] Step 5:

[0468] Compound Int-F-5 (250 mg, 0.25 mmol) was dissolved in dichloromethane (4 mL), and a hydrogen chloride / dioxane solution (4 M, 4 mL) was added at 0°C. The reaction system was stirred at 0°C for 2 hours. After completion of the reaction, the mixture was concentrated to afford compound Int-F-6 (220 mg, crude product), which was used directly in the next step.

[0469] MS m / z(ESI):710.5[M+H] + .

[0470] Step 6:

[0471] Crude compound Int-F-6 hydrochloride (220 mg, crude product from the previous step) was dissolved in dry N,N-dimethylformamide (4 mL). N,N-diisopropylethylamine (682 mg, 5.28 mmol) was added at 0°C, followed by compound Int-A-7 (660 mg, 0.88 mmol). The reaction was stirred at room temperature for 16 hours. After completion of the reaction, ethyl acetate (30 mL) was added, followed by washing with water (15 mL) and saturated sodium chloride solution (15 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified using a flash chromatography system (dichloromethane / methanol = 0-10%) to obtain compound Int-F-7 (220 mg, yield: 34.4%).

[0472] 1 H NMR (400MHz, CD3OD): δ5.97(s,3H),5.37-5.33(m,3H),4.99(d,3H),4.49-4.42(m,3H),4.37(d,3H),4.21(d,8H),4.08(d,3H),3.83(s,9H),3.7 3-3.57(m,40H),3.39-3.23(m,5H),3.19-3.12(m,3H),3.08-2.91(m,17 H),1.95-1.91(m,10H),1.53(s,27H),1.48(s,27H),1.39-1.34(m,18H).

[0473] Step 7:

[0474] Compound Int-F-7 (220 mg, 86.35 μmol) was dissolved in methanol (10 mL). A solution of lithium hydroxide monohydrate (22 mg, 0.52 mmol) in water (10 mL) was added at 0°C. The reaction was stirred at room temperature for 16 hours. After completion, IRN 77 resin was added for neutralization. The neutralized reaction solution was filtered and concentrated to afford compound Int-F-8 (190 mg, crude), which was used directly in the next reaction.

[0475] MS m / z(ESI):835.3([M+3H] / 3) + .

[0476] Step 8:

[0477] Compound Int-F-8 (190 mg, crude product from the previous step) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (4 mL) was added. The reaction was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, followed by the addition of water (4 mL) and stirring at room temperature for 1 hour. After concentration, the crude product was purified by high-performance liquid chromatography (HPLC column: SharpSil-T, 30*150 mm, 5 μm; mobile phase: aqueous phase (10 mM ammonium bicarbonate) and acetonitrile, gradient ratio: aqueous phase 25%-42%) to obtain the title product Int-F (53 mg, yield: 29.6%).

[0478] MS m / z(ESI):893.2([M+2H] / 2) + .

[0479] 1 H NMR (400MHz, CD3OD): δ5.92(d,3H),5.03(dd,3H),4.57(dd,3H),4.44(d,3H),4.31-4.19(m,5H),4.09-3 .99(m,4H),3.95-3.82(m,12H),3.82-3.46(m,46H),3.30-3.21(m,3H),2.98-2.93(m,10H),1.97(s,9H).

[0480] Example 7 Synthesis of Intermediate Int-G

[0481] Step 1:

[0482] To a solution of compound Int-F-1 (7.0 g, 23.86 mmol) in tetrahydrofuran (70 mL) was added a solution of lithium aluminum tetrahydride in tetrahydrofuran (2.5 M, 28.6 mL, 71.5 mmol) dropwise at 0°C. After the addition was complete, the mixture was heated to 70°C and stirred for 4 hours. After the reaction was complete, the cooled reaction solution was slowly poured into a solution of sodium sulfate decahydrate (40 g) in tetrahydrofuran (100 mL) at 0°C and stirred at room temperature for 1 hour. The solid was removed by filtration, and the mother liquor was directly concentrated under reduced pressure to obtain compound Int-G-2 (4.94 g, crude product), which was directly used in the next step.

[0483] MS m / z(ESI):208.1[M+H] + .

[0484] 1 H NMR (400MHz, CDCl3): δ3.73-3.71(m,2H),3.66-3.64(m,8H),3.62-3.58(m,4H),2.77-2.73(t,2H),2.42(s,3H).

[0485] Step 2:

[0486] To a solution of Int-G-2 (4.94 g, crude product from the previous step) in dichloromethane (60 mL) was added di-tert-butyl dicarbonate (6.31 g, 28.9 mmol) at 0°C and stirred overnight at room temperature. The reaction mixture was concentrated and then purified by silica gel column chromatography (dichloromethane / ethyl acetate = 10 / 1 to 2 / 1) to afford compound Int-G-3 (4.17 g, two-step yield: 56.9%).

[0487] MS m / z(ESI):330.2[M+Na] + .

[0488] 1 H NMR (400MHz, CDCl3): δ3.76-3.70(m,2H),3.70-3.54(m,12H),3.42-3.35(m,2H),2.91(s,3H),1.45(s,9H).

[0489] Step 3:

[0490] To a solution of Int-G-3 (4.0 g, 13.01 mmol) in dichloromethane (60 mL) at 0°C was added triphenylphosphine (5.11 g, 19.48 mmol). Stirring was continued for 10 minutes, followed by the addition of carbon tetrabromide (6.47 g, 19.51 mmol) in portions. The mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 to 2 / 1) to afford compound Int-G-4 (3.9 g, 81.0% yield).

[0491] 1 H NMR (400MHz, CDCl3): δ3.81(t,2H),3.71-3.55(m,10H),3.47(t,2H),3.39(s,2H),3.44-3.37(m,3H),1.45(s,9H).

[0492] Step 4:

[0493] To a solution of compound Int-C-4 (1.08 g, 2.48 mmol) in N,N-dimethylformamide (15 mL) were added compound Int-G-4 (3.22 g, 8.70 mmol) and potassium carbonate (1.71 g, 12.37 mmol), followed by heating and stirring at 65°C overnight. The reaction mixture was concentrated to remove N,N-dimethylformamide, and water (15 mL) was added. The mixture was extracted with dichloromethane (15 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-G-5 (1.29 g, 42.3% yield).

[0494] MS m / z(ESI):616.0([M+2H] / 2) + .

[0495] 1 H NMR (400MHz, CDCl3): δ4.21(d,2H),3.72-3.52(m,60H),3.43-3.35(m,8H),2.95-2.72(m,18H),1.45(s,27H).

[0496] Step 5:

[0497] To a solution of compound Int-G-5 (400 mg, 0.33 mmol) in dichloromethane (5.0 mL) was added a 4 M hydrogen chloride / dioxane solution (10 mL). The mixture was then stirred at room temperature for 2 hours and concentrated to afford compound Int-G-6 (360 mg, crude product), which was used directly in the next step.

[0498] MS m / z(ESI):465.9([M+2H] / 2) + .

[0499] Step 6:

[0500] To a solution of compound Int-G-6 (360 mg, crude product from the previous step) in N,N-dimethylformamide (10 mL) were added compound Int-A-7 (780 mg, 1.04 mmol) and N,N-diisopropylethylamine (418 mg, 3.23 mmol), followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated at room temperature to remove the N,N-dimethylformamide, and reverse-phase reaction was performed to afford compound Int-G-7 (500 mg, 55.6% yield over two steps).

[0501] MS m / z(ESI):692.8([M+4H] / 4) + .

[0502] 1H NMR (400MHz, CDCl3): δ11.40(s,3H),8.41(d,3H),6.15-6.09(m,3H),5.88(s,3H),5 .26-5.21(m,6H),4.43-4.34(m,6H),4.20(d,2H),4.17-3.99(m,10H),3.79(s,8H), 3.70-3.68(m,4H),3.70-3.68(m,46H),3.55-3.48(m,14H),3.41-3.28(m,4H),3.00 (s,4H),2.94(s,4H),2.76(s,10H),1.89(d,8H),1.48(d,54H),1.38-1.33(m,19H).

[0503] Step 7:

[0504] To a solution of compound Int-G-7 (200 mg, 72.25 μmol) in methanol (14.4 mL) was added an aqueous solution of lithium hydroxide monohydrate (30 mg, 0.71 mmol) (14.4 mL) at 0°C, followed by stirring at 20°C for 12 hours. After completion of the reaction, the reaction solution was adjusted to pH 7 using ion exchange resin IRN77. The filtered mother liquor was concentrated to afford compound Int-G-8 (197 mg, crude product), which was used directly in the next step.

[0505] MS m / z(ESI):682.3([M+4H] / 4) + .

[0506] Step 8:

[0507] To a solution of compound Int-G-8 (197 mg, crude product from the previous step) in dichloromethane (5.0 mL) was added trifluoroacetic acid (10 mL) at 0°C. The reaction was stirred at room temperature for 2 hours. After completion, the mixture was concentrated under reduced pressure, followed by the addition of water (4 mL) and stirring at room temperature for 1 hour. After concentration, the crude product was purified by HPLC (Waters XBridge, 19 x 150 mm, 5 μm; mobile phase A: 0.5% TFA in water, mobile phase B: acetonitrile, gradient: phase B 5% to 95%) to afford the title product, Int-G trifluoroacetate (25.0 mg, two-step yield: 13.4%).

[0508] 1H NMR(400MHz,D2O): δ5.59-5.57(m,3H),4.93-4.84(m,3H),4.43(dd,3H),4.32(dd,3H),4.16(s,2H),4.09-4.00(m,6H), 3.61-3.58(m,64H),3.41(dd,4H),3.31-3.28(m,2H),3.17-3.12(m,2H),2.89(s,4H),2.81-2.77(d,16H),1.88(s,9H).

[0509] Example 8 Synthesis of Intermediate Int-H

[0510] Step 1:

[0511] Compound Int-A-14 hydrochloride (572 mg, 1.46 mmol) was dissolved in dry N,N-dimethylformamide (10 mL), and potassium carbonate (2.50 g, 18.09 mmol) and compound Int-D-6 (2.30 g, 6.76 mmol) were added sequentially. The reaction was stirred at 65°C for 16 hours. After completion of the reaction, the reaction solution was concentrated, and water (30 mL) and dichloromethane (30 mL) were added. The aqueous phase was extracted with dichloromethane (30 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified using a flash chromatography elution system (dichloromethane / methanol = 0-10%) to obtain compound Int-H-1 (350 mg, yield: 22.7%).

[0512] MS m / z(ESI):1054.7[M+H] + .

[0513] 1 H NMR (400MHz, CDCl3): δ4.21(d,3H),3.72-3.49(m,45H),3.42-3.32(m,7H),2.91(s,9H),2.81-2.73(m,7H),2.45(s,1H),1.45(s,27H).

[0514] Step 2:

[0515] Compound Int-H-1 (250 mg, 0.24 mmol) was dissolved in dichloromethane (8 mL), and a hydrogen chloride / dioxane solution (4 M, 4 mL) was added at 0°C. The mixture was stirred at 0°C for 2 hours. After completion of the reaction, the mixture was concentrated to obtain compound Int-H-2 (222 mg, crude product), which was directly used in the next step.

[0516] MS m / z(ESI):754.5[M+H] + .

[0517] Step 3:

[0518] Compound Int-H-2 (222 mg, crude) was dissolved in dry N,N-dimethylformamide (5 mL). N,N-diisopropylethylamine (464 mg, 3.59 mmol) was added at 0°C, followed by compound Int-A-7 (600 mg, 0.80 mmol). The reaction was stirred at room temperature for 16 hours. After completion, ethyl acetate (30 mL) was added, followed by washing with water (15 mL) and saturated sodium chloride solution (15 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by flash chromatography using an eluent system (dichloromethane / methanol = 20:1 to 10:1) to obtain compound Int-H-3 (230 mg, two-step yield: 37.0%).

[0519] MS m / z(ESI):648.9([M+4H] / 4) + .

[0520] Step 4:

[0521] Compound Int-H-3 (230 mg, 88.74 μmol) was dissolved in methanol (14 mL), and a solution of lithium hydroxide monohydrate (30 mg, 0.71 mmol) in water (14 mL) was added at 0°C. The reaction was stirred at room temperature for 16 hours. After completion, IRN 77 resin was added for neutralization. The neutralized reaction solution was filtered and concentrated to afford compound Int-H-4 (226 mg, crude product), which was used directly in the next step.

[0522] MS m / z(ESI):850.7([M+3H] / 3) + .

[0523] Step 5:

[0524] The crude compound Int-H-4 (226 mg, crude product from the previous step) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (4 mL) was added. The reaction was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, followed by the addition of water (4 mL) and stirring at room temperature for 1 hour. After concentration, the crude product was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.5% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain the title product, Int-H trifluoroacetate (86.8 mg, two-step yield: 40.7%).

[0525] MS m / z(ESI):610.5([M+3H] / 3) + .

[0526] 1 H NMR (400MHz, CD3OD): δ5.91(dd,3H),5.06-4.99(m,3H),4.57(dd,3H),4.44(d,3H),4.28-4.18(m,5H),4 .12-3.99(m,4H),3.99-3.79(m,14H),3.79-3.47(m,48H),3.23-3.14(m,3H),2.97(d,10H),1.97(d,9H).

[0527] Example 9 Synthesis of Intermediate Int-I

[0528] Step 1:

[0529] Compound Int-B-3 (900 mg, 2.59 mmol) was dissolved in dry N,N-dimethylformamide (10 mL), and potassium carbonate (2.6 g, 18.81 mmol) and compound Int-D-6 (2.50 g, 7.66 mmol) were added sequentially. The reaction was stirred at 65°C for 16 hours. After completion of the reaction, the reaction solution was concentrated, and water (30 mL) and dichloromethane (30 mL) were added. The aqueous phase was extracted with dichloromethane (30 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by flash chromatography using an elution system (dichloromethane / methanol = 20 / 1 to 10 / 1) to obtain compound Int-I-1 (300 mg, yield: 11.5%).

[0530] MS m / z(ESI):1032.7[M+Na] + .

[0531] 1 H NMR (400MHz, CDCl3): δ4.20(d,2H),3.86-3.51(m,40H),3.42-3.16(m,16H),2.89(d,9H),2.47(t,1H),1.45(s,27H).

[0532] Step 2:

[0533] Compound Int-I-1 (300 mg, 0.30 mmol) was dissolved in dichloromethane (8 mL), and a hydrogen chloride / dioxane solution (4 M, 4 mL) was added at 0°C. The reaction was stirred at 0°C for 2 hours. After completion of the reaction, the mixture was concentrated to obtain compound Int-I-2 hydrochloride (265 mg, crude product), which was used directly in the next reaction.

[0534] MS m / z(ESI):710.5[M+H] + .

[0535] Step 3:

[0536] Compound Int-I-2 (265 mg, crude) was dissolved in dry N,N-dimethylformamide (8 mL). N,N-diisopropylethylamine (374 mg, 2.89 mmol) was added at 0°C, followed by compound Int-A-7 (750 mg, 1 mmol). The reaction was stirred at room temperature for 16 hours. After completion of the reaction, ethyl acetate (50 mL) was added, followed by washing with water (30 mL) and saturated sodium chloride solution (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified using a flash elution system (dichloromethane / methanol = 0-10%) to obtain compound Int-I-3 (330 mg, two-step yield: 43.2%).

[0537] 1 H NMR (400MHz, CDCl3): δ11.41(s,3H),8.41(d,3H),5.95-5.81(m,6H),5.30-5.21(m,6H),4.43-4.34(m,6H),4.20(d,2H),4.10-3.95(m,6H),3. 79(s,9H),3.72-3.49(m,42H),3.40-3.10(m,8H),2.97(d,9H),2.78-2. 60(m,9H),2.46(s,1H),1.90(d,9H),1.48(d,54H),1.39-1.33(m,18H).

[0538] Step 4:

[0539] Compound Int-I-3 (330 mg, 0.13 mmol) was dissolved in methanol (20 mL), and a 20 mL aqueous solution of lithium hydroxide monohydrate (42 mg, 1.00 mmol) was added at 0°C. The reaction was stirred at room temperature for 16 hours. After completion of the reaction, IRN 77 resin was added for neutralization. The neutralized reaction solution was filtered and concentrated to afford compound Int-I-4 (285 mg, crude product), which was used directly in the next reaction.

[0540] MS m / z(ESI):836.1([M+3H] / 3) + .

[0541] Step 5:

[0542] Compound Int-I-4 (285 mg, crude product) was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (6 mL) was added. The reaction was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, followed by the addition of water (4 mL) and stirring at room temperature for 1 hour. After concentration, the crude product was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.5% TFA in water, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain the title product, Int I trifluoroacetate (154.0 mg, two-step yield: 50.3%).

[0543] 1 H NMR (400MHz, CD3OD): δ5.94(d,3H),5.08-4.99(m,3H),4.57(d,3H),4.44(d,3H),4.30-4.19(m,5H),4. 08-4.00(m,4H),3.92-3.77(m,10H),3.73-3.46(m,48H),3.24-3.14(m,3H),2.98(d,10H),1.97(d,9H).

[0544] Example 10 Synthesis of Intermediate Int-J

[0545] Step 1:

[0546] To a solution of compound Int-A-14 (900 mg, 2.30 mmol) in N,N-dimethylformamide (15 mL) were added compound Int-G-4 (3.35 g, 9.05 mmol) and potassium carbonate (3.89 g, 28.15 mmol), followed by heating and stirring at 65°C overnight. The reaction mixture was concentrated to remove N,N-dimethylformamide, and water (15 mL) was added. The mixture was extracted with dichloromethane (15 mL x 3). The combined organic phases were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1 to 10 / 1) to afford compound Int-J-1 (1.20 g, 44.0% yield).

[0547] MS m / z(ESI):594.0([M+2H] / 2) + .

[0548] 1H NMR (400MHz, CDCl3): δ4.20(d,2H),3.76-3.51(m,54H),3.43-3.32(m,6H),2.91(s,9H),2.90-2.78(m,9H),2.29(s,4H),1.45(s,27H).

[0549] Step 2:

[0550] To a solution of compound Int-J-1 (400 mg, 0.34 mmol) in dichloromethane (10 mL) was added a 4 M hydrogen chloride / dioxane solution (10 mL) at 0°C. The mixture was then stirred at room temperature for 2 hours and concentrated to afford compound Int-J-2 (360 mg, crude product), which was used directly in the next reaction.

[0551] MS m / z(ESI):908.8[M+Na] + .

[0552] Step 3:

[0553] To a solution of compound Int-J-2 (360 mg, crude product from the previous step) in N-dimethylformamide (10 mL) were added Int-A-7 (809 mg, 1.08 mmol) and N,N-diisopropylethylamine (433 mg, 3.35 mmol), followed by stirring at room temperature for 12 hours. The reaction mixture was concentrated at room temperature to remove the N,N-dimethylformamide, and the mixture was reversed to afford compound Int-J-3 (640 mg, 69.1% yield over two steps).

[0554] MS m / z(ESI):681.9([M+4H] / 4) + .

[0555] Step 4:

[0556] To a solution of compound Int-J-3 (600 mg, 0.22 mmol) in methanol (44 mL) was added a solution of lithium hydroxide (92.4 mg, 2.20 mmol) (44 mL) at 0°C, followed by stirring at 20°C for 12 hours. After completion of the reaction, the reaction solution was adjusted to pH 7 using ion exchange resin IRN77. The filtered mother liquor was concentrated to afford compound Int-J-4 (590 mg, crude product), which was used directly in the next reaction.

[0557] MS m / z(ESI):671.0([M+4H] / 4) + .

[0558] Step 5:

[0559] To a solution of compound Int-J-4 (590 mg, crude product from the previous step) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) at 0°C. The mixture was then stirred at 20°C for 2 hours, concentrated, and then water (10 mL) was added and stirred for 2 hours. The mixture was concentrated under reduced pressure, and the crude product was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.5% TFA in water, mobile phase B: acetonitrile, gradient: phase B 5% to 95%) to afford the title product, Int-J trifluoroacetate (140 mg, two-step yield: 25.1%).

[0560] 1 H NMR(400MHz,D2O): δ5.52(d,3H),4.85-4.75(m,3H),4.37(dd,3H),4.26(dd,3H),4.09(s,2H),4.02-3.9 4(m,6H),3.52(br,62H),3.35(dd,3H),3.31-2.97(m,4H),2.88(s,4H),2.75-2.71(m,15H),1.81(s,9H).

[0561] Example 11 Synthesis of Intermediate Int-K

[0562] Step 1:

[0563] Compound Int-B-3 (586 mg, 1.69 mmol) was dissolved in dry N,N-dimethylformamide (10 mL), and potassium carbonate (3.0 g, 21.71 mmol) and compound Int-G-4 (3.20 g, 8.64 mmol) were added sequentially. The reaction was stirred at 65°C for 16 hours. After completion of the reaction, the reaction solution was concentrated, and water (30 mL) and dichloromethane (30 mL) were added. The aqueous phase was extracted with dichloromethane (30 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified using a flash prep system (dichloromethane / methanol = 0-10%) to obtain compound Int-K-1 (800 mg, yield: 41.4%).

[0564] MS m / z(ESI):1165.7[M+Na] + .

[0565] Step 2:

[0566] Compound Int-K-1 (300 mg, 0.26 mmol) was dissolved in dichloromethane (8 mL), and a hydrogen chloride / dioxane solution (4 M, 4 mL) was added at 0°C. The mixture was stirred at 0°C for 2 hours. After completion of the reaction, the mixture was concentrated to obtain compound Int-K-2 (269 mg, crude product), which was used directly in the next reaction.

[0567] MS m / z(ESI):421.9([M+2H] / 2) + .

[0568] Step 3:

[0569] Compound Int-K-2 (269 mg, crude product from the previous step) was dissolved in dry N,N-dimethylformamide (6 mL). N,N-diisopropylethylamine (373 mg, 2.89 mmol) was added under ice-cooling, followed by compound Int-A-7 (650 mg, 0.86 mmol). The reaction was stirred at room temperature overnight. After completion, ethyl acetate (30 mL) was added, followed by washing with water (15 mL) and saturated sodium chloride solution (15 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified using a flash prep system (dichloromethane / methanol = 0-10%) to obtain compound Int-K-3 (350 mg, two-step yield: 50.2%).

[0570] MS m / z(ESI):894.1([M+3H] / 3) + .

[0571] 1 H NMR (400MHz, CDCl3): δ11.39(s,3H),8.41(d,3H),6.36-6.12(m,3H),5.90(dd,3H),5.36-5.16(m,6H),4.43-4.35(m,6H),4.23-3.9 5(m,14H),3.80-3.58(m,62H),3.41-3.17(m,12H),3.01-2.98(m,9H),2.50(t,1H),1.89(s,9H),1.48(d,54H),1.38-1.33(m,18H).

[0572] Step 4:

[0573] Compound Int-K-3 (350 mg, 0.13 mmol) was dissolved in methanol (22 mL). A 22 mL solution of lithium hydroxide monohydrate (45 mg, 1.07 mmol) was added under ice. The reaction was stirred at room temperature for 16 hours. After completion, IRN 77 resin was added for neutralization. The neutralized reaction solution was filtered and concentrated to afford compound Int-K-4 (310 mg, crude product), which was used directly in the next reaction.

[0574] MS m / z(ESI):880.2([M+3H] / 3) + .

[0575] Step 5:

[0576] Compound Int-K-4 (310 mg, crude product from the previous step) was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (6 mL) was added. The reaction was stirred at room temperature for 2 hours. After completion, the mixture was concentrated under reduced pressure, followed by the addition of water (4 mL) and stirring at room temperature for 1 hour. After concentration, the crude product was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.5% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain the title product, Int-K trifluoroacetate (203 mg, two-step yield: 62.9%).

[0577] MS m / z(ESI):639.8([M+3H] / 3) + .

[0578] 1 H NMR (400MHz, CD3OD): δ5.94(s,3H),5.03(t,3H),4.58(dd,3H),4.42(dd,3H),4.29-4.19(m,5H),4.13-4.01(m,3H) ,3.90-3.76(m,10H),3.74-3.58(m,51H),3.55-3.44(m,10H),3.26-3.17(m,3H),3.03-2.94(m,10H),1.97(s,9H).

[0579] Example 12 Synthesis of Intermediate Int-L

[0580] Step 1: Synthesis of Int-L-2

[0581] Compound Int-L-1 (0.176 g, 0.19 mmol, prepared using a known method, Journal of Organic Chemistry, 2012, vol. 77, #20, pp. 8879-8887) was dissolved in 10 mL of THF, and 10% Pd (0.124 g) was added. The air in the system was replaced with hydrogen four times, and the mixture was allowed to react at room temperature under a hydrogen atmosphere for four hours. After completion of the reaction, the mixture was filtered and concentrated to yield 0.16 g of crude product, which was used directly in the next step.

[0582] MS m / z(ESI):830.5[M+H] + .

[0583] Step 2: Synthesis of compound Int-L-3

[0584] Compound Int-L-2 (0.16 g, 0.19 mmol) was dissolved in dry tetrahydrofuran (4 mL), and a solution of potassium tert-butoxide in THF (0.39 mL, 0.394 mmol) was added under ice-water cooling. After the addition, the reaction was stirred at room temperature for half an hour. The mixture was then cooled in an ice-water bath, and propyne bromide (0.0703 g, 0.591 mmol) was added. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was filtered, and the filter cake was washed with DCM, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 1:5) to afford 0.134 g of compound Int-L-3 (yield: 81%).

[0585] 1 H NMR (400MHz, CDCl3): δ4.60(t,3H),4.17(d,2H),3.87~3.44(m,46H),3.40(s,8H),2.42(t,1H),1.86~1.44(m,18H).

[0586] Step 3: Synthesis of compound Int-L-4

[0587] Compound Int-L-3 (134 mg, 0.154 mmol) was dissolved in anhydrous methanol (5 mL), and p-toluenesulfonic acid monohydrate (14 mg, 0.072 mmol) was added. The reaction was stirred at room temperature for 1 hour. After completion of the reaction, sodium bicarbonate (20 mg, 0.238 mmol) was added for neutralization, and the mixture was concentrated to obtain 95 mg of crude product, which was used directly in the next step.

[0588] MS m / z(ESI):615.4[M+H] + .

[0589] Step 4: Synthesis of compound Int-L-5

[0590] Compound Int-L-4 (95 mg, 0.154 mmol) was dissolved in DCM (5 mL), and triethylamine (156 mg, 1.542 mmol), p-toluenesulfonyl chloride (147 mg, 0.771 mmol), and DMAP (0.9 mg, 0.008 mmol) were added sequentially. The reaction was stirred at room temperature overnight. After completion, saturated aqueous sodium bicarbonate was added to quench the reaction, followed by extraction with DCM, drying, filtration, and concentration to obtain 166 mg of crude product, which was used directly in the next step.

[0591] MS m / z(ESI):1077.3[M+H] + .

[0592] Step 5: Synthesis of compound Int-L-6

[0593] Compound Int-L-5 (166 mg, 0.154 mmol) was dissolved in ethanolic methylamine (5 mL, approximately 30%) and stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated, and the concentrate was dissolved in water and DCM. The organic layer was separated, and the aqueous layer was extracted with DCM. The aqueous phase was concentrated under reduced pressure to provide 70 mg of compound Int-L-6 (yield: 70%).

[0594] MS m / z(ESI):654.4[M+H] + .

[0595] Step 6: Synthesis of compound Int-L-7

[0596] To a solution of compound Int-L-6 (193 mg, 0.295 mmol) in N,N-dimethylformamide (8 mL) and dichloromethane (8 mL) were added compound Int-A-7 (765 mg, 1.02 mmol) and N,N-diisopropylethylamine (571 mg, 4.43 mmol), followed by heating to 60°C and stirring for 5 hours. The reaction solution was concentrated at room temperature to remove dichloromethane and N,N-dimethylformamide, and purified by column chromatography (acetonitrile:water = 0-100%) to afford 322 mg of compound Int-L-7 (yield: 44%).

[0597] MS m / z(ESI):831.4([M / 3+H] + .

[0598] 1H NMR (400MHz, CDCl3): δ11.41(bs,2H),8.75(bs,2H),6.76(bs,2H),5.86(s,3H),5.50-5.22(m,6H),4.43-4.38(m,6H),4.21-3 .98(m,11H),3.80(s,9H),3.69-3.23(m,48H),2.99-2.94(m,9H),2.48(s,1H),1.85(s,9H),1.50(m,52H),1.40-1.30(m,23H).

[0599] Step 7: Synthesis of compound Int-L-8

[0600] To a solution of compound Int-L-7 (260 mg, 0.104 mmol) in methanol (4.2 mL) was added an aqueous solution (4.2 mL) of lithium hydroxide monohydrate (26 mg, 0.627 mmol) at 0°C, followed by stirring at 20°C for 10 minutes. After the reaction, the reaction solution was adjusted to pH 5-6 using ion exchange resin IRN77, and the filtered mother liquor was concentrated to obtain 250 mg of compound Int-L-8 (yield: 98%).

[0601] MS m / z(ESI):1225.5([M / 2+H] + .

[0602] Step 8: Synthesis of compound Int-L

[0603] To a solution of compound Int-L-8 (250 mg, 0.102 mmol) in dichloromethane (2.1 mL) was added trifluoroacetic acid (2.1 mL) at 0°C. The reaction was stirred at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, followed by addition of acetonitrile (2.1 mL) and water (2.1 mL), and stirred at room temperature for 3 hours. The reaction solution was concentrated and purified by column chromatography (TFA:acetonitrile = 5% to 95%) to obtain 65 mg of compound Int-L (TFA salt, yield: 37%).

[0604] 1 H NMR (400MHz, DMSO-d6): δ12.87(bs,2H),8.19-8.03(m,3H),7.98-7.00(m,14H),5.71(s,3H),5.22-4.82(m,6H),4.62 -4.29(m,6H),4.20(d,2H),4.14-3.81(m,6H),3.70-3.68(m,48H),3.55-3.48(m,9H),2.94-2.85(m,9H),1.75(s,9H).

[0605] MS m / z(ESI):865.1([M / 2+H] + .

[0606] Example 13 Synthesis of Lysine-Conjugated Conjugate 1-D-E1

[0607] Preparation of Azido-PEG4-NHS ester solution: 16.75 mg of PEG4-azido NHS (CAS: 944251-24-5, purchased from Shaanxi Xinyan Bomei Biological) was dissolved in 100 μl of DMF and then diluted with 1× PBS pH 7.2 buffer solution at 0°C to a final concentration of 50 mM.

[0608] Prepare click reagent: dissolve 10.0 mg of CuSO4 (5 mM) in 12.53 ml of 1× PBS, take 5 ml and add it to 43.1 mg of BTTAA (20 mM, CAS: 1334179-85-9, purchased from Anage) and 247.5 mg of sodium ascorbate (250 mM) to dissolve for later use.

[0609] Step 1: To a 15 mL reaction tube, add protein Fc (963.4 μL, 10.38 mg / mL, sequence shown in SEQ ID NO: 1), PBS pH 7.2 (36.6 μL), and PEG4-azido solution (25.0 μL). Shake at room temperature for 3 hours. Purify the product using a centrifugal desalting column in PBS pH 6.3 to obtain Fc-azido (8.95 mg, 89.5% yield, 97.8% purity). Maldi ion analysis revealed a DAR value of 5.8.

[0610] Step 2: To a 15 mL reaction tube, add Fc-azido (8.95 mg), intermediate compound Int-D (4.49 mg) in 1×PBS pH 6.3 (76 μL), and click reagent (499 μL). Shake at room temperature for 20 hours. Desalt the mixture with PBS (pH 5.0) and purify it by column to obtain conjugate 1-D-E1 (6.9 mg, 86.4% yield, 98.2% purity). Maldi-TOF analysis revealed a DAR value of 3.86.

[0611] Example 14 Synthesis of Lysine-Conjugated Conjugate 1-L-E1

[0612] Step 1: Fc-azido (8.81 mg, yield 88.1%, purity 97.7%) was prepared using the same method as in Example 13. Maldi tof analysis showed a DAR value of 5.57.

[0613] Step 2: To a 15 mL reaction tube, add Fc-azido (8.81 mg), intermediate compound Int-L (4.07 mg) in 1×PBS pH 6.3 (313 μL), and click reagent (490 μL). Shake at room temperature for 20 hours. Desalt with PBS (pH 5.0) and purify by column to obtain conjugate 1-L-E1 (6.6 mg, 74.9% yield, 98.8% purity). Maldi-TOF analysis revealed a DAR value of 3.98.

[0614] Example 15 Synthesis of conjugate molecules based on interchain disulfide bond cysteine ​​coupling

[0615] Purified Fc was exchanged into 0.1 M phosphate buffer (pH 8.0) containing 2 mM EDTA using a 30,000 Dalton molecular weight cutoff centrifuge concentrator to a final concentration of 2.5 mg / ml. DTT (Biyuntian) was added to the 2.5 mg Fc solution at a final concentration of 3 mM, mixed, and incubated at 37°C for 2-3 hours. After completion of the reaction, excess DTT was removed using Sephadex G-25 resin (Cytiva). The reduced cysteine ​​content in the solution was determined using DTNB (Solarbio).

[0616] To 2.5 mg of reduced Fc, add 10 equivalents of Azido-PEG3-Maleimide (purchased from Alfa Aesar, dissolved in a 0.01 M stock solution in DMSO / PBS). Mix thoroughly and incubate at room temperature with shaking for 2-3 hours to produce Fc-azido. Immediately thereafter, add 20 equivalents of free cysteine ​​to quench unreacted Azido-PEG3-Maleimide, and incubate at 10°C for 30 minutes. After completion of the reaction, exchange the buffer with PBS using Sephadex G-25 resin. Add 10 equivalents of the aforementioned intermediate to Fc-azido, mix thoroughly, and then add 10 equivalents of a copper ion catalyst (0.005 M copper sulfate, 0.02 M BTTAA, and 0.25 M sodium ascorbate) to initiate the click reaction. Incubate at room temperature for 12-20 hours. After the reaction was completed, the title conjugate molecule was purified using a MabPurix prepacked column and HiLoad Superdex 200 16 / 600PG, and the conjugate molecule was analyzed for its DAR value.

[0617] Example 16 Synthesis of conjugate molecules based on single-site cysteine ​​mutation conjugation

[0618] Purified Fc single-site cysteine ​​mutants were replaced with 2 mM EDTA in 0.1 M phosphate buffer (pH 8.0) using a 30,000 Dalton molecular weight cutoff centrifugal concentrator to a final concentration of 2.5 mg / ml. DTT was added to 2.5 mg of the Fc mutant to a final concentration of 3 mM, mixed, and incubated at 37°C for 2-3 hours. After completion of the reaction, excess DTT was removed through Sephadex G-25 resin. The reduced cysteine ​​content in the solution was determined using DTNB. The reduced Fc mutants were replaced with 2 mM EDTA and 1 mM dehydroascorbic acid in 0.1 M phosphate buffer (pH 6.5) using Sephadex G-25 resin. After incubation at room temperature for 2-3 hours, the Fc mutants were replaced with 2 mM EDTA in 0.1 M phosphate buffer (pH 8.0) using Sephadex G-25 resin. The content of reduced cysteine ​​in the solution was determined using DTNB.

[0619] To 2.5 mg of the heavily oxidized Fc mutant, add 5 equivalents of Azido-PEG3-Maleimide (0.01 M stock solution in DMSO / PBS), mix thoroughly, and incubate at room temperature with shaking for 2-3 hours. Immediately thereafter, add 20 equivalents of free cysteine ​​to quench unreacted Azido-PEG3-Maleimide, and incubate at 10°C for 30 minutes. After completion of the reaction, exchange the buffer with PBS using Sephadex G-25 resin. Add 5 equivalents of the aforementioned intermediate to the Fc mutant-azido, mix thoroughly, and then add 10 equivalents of a copper ion catalyst (0.005 M copper sulfate, 0.02 M BTTAA, and 0.25 M sodium ascorbate) to initiate the click reaction. Incubate at room temperature for 12-20 hours. After the reaction was completed, the conjugate molecule was purified using a MabPurix prepacked column and HiLoad Superdex 200 16 / 600PG and its DAR value was analyzed.

[0620] Example 17 Synthesis of conjugate molecules based on double-point cysteine ​​mutation conjugation

[0621] The purified Fc double-site cysteine ​​mutant was replaced with 2 mM EDTA in 0.1 M phosphate buffer (pH 8.0) using a 30,000 Dalton molecular weight cutoff centrifugal concentrator to a final concentration of 2.5 mg / ml. DTT was added to 2.5 mg of the Fc mutant to a final concentration of 3 mM, mixed, and incubated at 37°C for 2-3 hours. After completion of the reaction, excess DTT was removed by Sephadex G-25 resin. The reduced cysteine ​​content in the solution was determined using DTNB. The reduced Fc mutant was replaced with 2 mM EDTA and 1 mM dehydroascorbic acid in 0.1 M phosphate buffer (pH 6.5) using Sephadex G-25 resin. After incubation at room temperature for 2-3 hours, the Fc mutant was replaced with 2 mM EDTA in 0.1 M phosphate buffer (pH 8.0) using Sephadex G-25 resin. The content of reduced cysteine ​​in the solution was determined using DTNB.

[0622] To 2.5 mg of the heavily oxidized Fc mutant, add 10 equivalents of Azido-PEG3-Maleimide (0.01 M stock solution in DMSO / PBS), mix thoroughly, and incubate at room temperature with shaking for 2-3 hours. Immediately thereafter, add 20 equivalents of free cysteine ​​to quench unreacted Azido-PEG3-Maleimide, and incubate at 10°C for 30 minutes. After completion of the reaction, exchange the buffer with PBS using Sephadex G-25 resin. Add 10 equivalents of the aforementioned intermediate to the Fc mutant-azido, mix thoroughly, and then add 10 equivalents of a copper ion catalyst (0.005 M copper sulfate, 0.02 M BTTAA, and 0.25 M sodium ascorbate) to initiate the click reaction. Incubate at room temperature for 12-20 hours. After the reaction was completed, the conjugate molecule was purified using a MabPurix prepacked column and HiLoad Superdex 200 16 / 600PG and its DAR value was analyzed.

[0623] Example 18 Synthesis of Conjugate 2-D-E1 Based on Sugar Site-Directed Conjugation

[0624] Prepare click reagent: dissolve 10.0 mg of CuSO4 (5 mM) in 12.53 ml of 1× PBS, take 5 ml and add it to 43.1 mg of BTTAA (20 mM, CAS: 1334179-85-9, purchased from Anage) and 247.5 mg of sodium ascorbate (250 mM) to dissolve for later use.

[0625] Step 1: Fc (963.4 μL, 10.38 mg / mL), 1× PBS pH 7.2 (36.6 μL), and Endos (WT) (0.1 mg, 122 μL, 0.82 mg / mL) were added to a 15 mL reaction tube and shaken at 37°C for 3 hours. N3-PEG3-Oxa (3.11 mg) in 1× PBS pH 7.2 (30 μL) was added and shaken at 37°C for 1 hour. After completion of the reaction, 9.5 mg of the conjugate Fc-1 was purified using a MabPurix prepacked column and a HiLoad Superdex 200 16 / 600PG column to obtain 95.0% yield, 98.5% purity with a DAR value of 1.82.

[0626] Step 2: To a 15 mL reaction tube, add Fc-1 (9.5 mg), intermediate compound Int-D (3.39 mg) in 1×PBS pH 6.3 (76 μL), and click reagent (529 μL). Shake at room temperature for 20 hours. Desalt with PBS (pH 5.0) and purify by column to obtain conjugate 2-D-E1 (7.5 mg, 78.9% yield, 98.0% purity). Maldi-TOF analysis revealed a DAR value of 3.80.

[0627] Example 19 Synthesis of Intermediate Int-L1

[0628] Step 1: Synthesis of Int-L1-3

[0629] Under nitrogen protection, compound Int-L1-1 (0.35 g, 1.20 mmol) and 2,6-difluoro-3-hydroxypyridine (0.2 g, 1.56 mmol) were dissolved in DCM (5 mL). EDCI (0.30 g, 1.56 mmol) was added under ice-water cooling, and the reaction was then heated to room temperature and stirred for 2 h. The reaction mixture was directly concentrated and purified on a reverse phase column (0.1% TFA in H2O:ACN = 60:40) to give 0.48 g of Int-L1-3, yield: 98%.

[0630] MS m / z(ESI):427.1[M+Na].

[0631] Step 2: Synthesis of Int-L1

[0632] Under nitrogen protection, compound Int-L (TFA salt, 0.32 g, 0.139 mmol) and Int-L1-3 (0.23 g, 0.556 mmol) were dissolved in NMP (1 mL), and copper tetraacetonitrile hexafluorophosphate (0.16 g, 0.418 mmol) was added at room temperature, followed by reaction at room temperature for 20 minutes; the product was directly sent for preparative purification by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain 0.2 g of the TFA salt of compound Int-L1, with a yield of 53.7% and a purity of 93.9%.

[0633] 1 H NMR (400MHz, D2O): δ8.02(s,1H),7.90-7.82(m,1H),7.01-6.98(m,1H),5.87(s,3H),4.92-4.89(m,5H),4.59-4.48(m,7H),4.36(d,3 H),4.13-3.98(m,6H),3.89-3.81(m,5H),3.75-3.51(m,51H),3.46-3.38(m,11H),3.32-3.13(m,3H),2.93-2.74(m,9H),1.89(s,9H).

[0634] MS m / z(ESI):1067.3[M / 2+H] + .

[0635] Example 20 Synthesis of Intermediate Int-M

[0636] Step 1: Synthesis of Int-M-2

[0637] KOH (756 mg, 13.5 mmol) was added to a 25 mL single-necked bottle at room temperature, and DMSO (anhydrous, 5 ml) was added and stirred evenly; compound Int-G-3 (1.38 g, 4.5 mmol) was added and stirred at room temperature for 15 minutes; Int-M-1 (591 mg, 1.0 mmol, prepared by a known method, Journal of Organic Chemistry) was added. Chemistry, 2012, vol. 77, # 20, p. 8879–8887) and KI (83 mg, 0.5 mmol) were stirred for 10 minutes; the temperature was raised to 75°C for reaction for 6 hours; the mixture was cooled to room temperature, diluted with 20 mL of ethyl acetate, and 30 mL of deionized water was slowly added under an ice-water bath. The aqueous phase was extracted twice with ethyl acetate (2×20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to obtain a crude product; the crude product was purified by reverse phase column chromatography (ACN:H2O=90%) to obtain 1.0 g of compound Int-M-2 with a yield of 78.7% and a purity of 95%.

[0638] MS m / z(ESI):1293.7[M+Na].

[0639] Step 2: Synthesis of compound Int-M-3

[0640] Compound Int-M-2 (1.0 g, 0.787 mmol) and 20% Pd(OH)2 / C (0.8 g) were added to a 50 mL single-necked flask, followed by the addition of MeOH (30 mL) and stirring at room temperature for 5 minutes; the mixture was then replaced three times under an atmospheric pressure of hydrogen, heated to 30°C, and reacted for 18 hours; filtered through celite, and the filter cake was washed three times with methanol (10 mL × 3). The filtrate was directly concentrated to obtain 0.9 g of crude compound Int-M-3 with a yield of 106.06%, which was used directly in the next step.

[0641] MS m / z(ESI):1198.7[M+H2O].

[0642] Step 3: Synthesis of compound Int-M-4

[0643] Compound Int-M-3 (0.9 g, 0.762 mmol) was dissolved in anhydrous THF (10 mL), cooled to 0° C. in an ice-water bath, and stirred; potassium tert-butoxide (1.54 mL, 1.54 mmol, 1 M in THF) was added, then the mixture was warmed to room temperature and stirred for one hour; ice-cooled again, 3-bromopropyne (272 mg, 2.29 mmol) was added dropwise, and the mixture was slowly warmed to room temperature and reacted for two hours; ice-cooled, the reaction solution was quenched with saturated aqueous ammonium chloride (30 mL), and then extracted with EtOAc (30 mL×3); the organic phases were combined, washed with water (20 mL) and saturated brine (20 mL) in turn, dried over anhydrous sodium sulfate (30 g), filtered, and dried under reduced pressure. The resulting residue was purified by reverse phase column chromatography (ACN: 0.1% TFA in H2O=50%-75%) to obtain 0.8 g of compound Int-M-4 with a purity of 80% and a yield of 86%.

[0644] MS m / z(ESI):1118.4[M-100].

[0645] Step 4: Synthesis of compound Int-M-5

[0646] Compound Int-M-4 (0.9 g, 0.75 mmol) was dissolved in DCM (11 mL), cooled in an ice-water bath, stirred, and then TFA (11 mL) was added dropwise. The reaction was allowed to warm to room temperature and reacted for two hours. The reaction was stopped and concentrated directly. The residue was washed twice with DCM (10 mL x 2) and then pumped dry with an oil pump to obtain the crude compound Int-M-5, which was used directly in the next step.

[0647] MS m / z(ESI):459.8[M / 2+H] + .

[0648] Step 5: Synthesis of compound Int-M-6

[0649] Compound Int-M-5 (350 mg, 0.382 mmol) was added to a single-necked flask, DCM (6 mL) and DMF (6 mL) were added at room temperature, DIEA (0.99 g, 7.65 mmol) was added, and the mixture was stirred at room temperature for 5 minutes; compound Int-A-7 (0.862 g, 1.15 mmol) was added in batches, replaced three times under nitrogen protection, and then heated to 49 ° C for 10 hours. The reaction was stopped and concentrated directly. The residue was dissolved in H2O (20 mL) and extracted with EA (20 mL × 2). The organic phases were combined, concentrated under reduced pressure, and then purified by reverse phase column (ACN / H2O, 90-93% product) to obtain 0.8 g of compound Int-M-6, yield: 75.9%, purity: 84%.

[0650] MS m / z(ESI):919.5[M / 3+H] + .

[0651] Step 6: Synthesis of compound Int-M-7

[0652] Compound Int-M-6 (0.8 g, 0.29 mmol) was dissolved in MeOH (7 mL) and THF (7 mL), cooled in an ice-water bath, and H₂O (7 mL) was added, followed by lithium hydroxide monohydrate (88 mg, 2.32 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 3 hours. A cation exchange resin was added until the pH reached 5-6. The mixture was then filtered, and the filter cake was washed with MeOH (10 mL x 2). The filtrate was concentrated to afford 0.6 g of compound Int-M-7, a yield of 76.2%. This was carried on to the next step.

[0653] MS m / z(ESI):905.3[M / 3+H] + .

[0654] Step 7: Synthesis of compound Int-M-8

[0655] Compound Int-M-7 (0.6 g, 0.224 mmol) was dissolved in DCM (4 mL), TFA (4 mL) was added under ice-water cooling, and the reaction was then warmed to room temperature and stirred for 2 hours; the residue was directly concentrated, and the residue was washed twice with dichloromethane (4 mL), then dissolved in ACN (5 mL) and H2O (5 mL), and stirred at room temperature for 3 hours; the residue was directly concentrated and purified by reverse phase column (ACN / 0.1% TFA in H2O, 40% product) to obtain 0.42 g of TFA salt of compound Int-M-8, yield: 73%, purity: 87.6%.

[0656] MS m / z(ESI):997.3[M / 2+H] + .

[0657] Step 8: Synthesis of compound Int-M

[0658] Under nitrogen protection, compound Int-M-8 (TFA salt, 50 mg, 0.025 mmol) and compound Int-L1-3 (55 mg, 0.15 mmol) were dissolved in NMP (1 mL), and copper tetraacetonitrile hexafluorophosphate (27 mg, 0.075 mmol) was added at room temperature, followed by reaction at room temperature for 20 minutes; the product was directly sent for preparative purification by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to give 35 mg of compound Int-M as a TFA salt, with a yield of 58% and a purity of 92.8%.

[0659] 1 H NMR (400MHz, D2O): δ7.99(s,1H),7.90-7.82(m,1H),7.02-6.98(m,1H),5.77(s,3H),4.92-4.88(m,5H),4.59-4.46(m,13H),4.35(d,5H),4.1 3-3.98(m,10H),3.89-3.81(m,6H),3.68-3.50(m,47H),3.46-3.39(m, 11H),3.30-3.15(m,11H),2.93-2.88(m,7H),2.81(s,6H),1.89(s,9H).

[0660] MS m / z(ESI):1199.4[M / 2+H] + .

[0661] Example 21 Synthesis of Intermediate Int-N

[0662] Step 1: Synthesis of compound Int-N-2

[0663] In an ice-water bath, compound Int-M-1 (2.2 g, 3.72 mmol, prepared by a known method, Journal of Organic Chemistry) was added. Chemistry, 2012, vol. 77, #20, p. 8879–8887) was added to a 50 mL single-necked flask, and DMAc (anhydrous, 10 mL) was added and stirred evenly. tBuOK (477 mg, 5.0 mmol) was added and stirred for 10 minutes. Compound Int-N-1 (purchased from Leyan, 0.69 g, 2.47 mmol) was added, and the temperature was raised to 60°C for 16 hours. The mixture was cooled to room temperature and diluted with 20 mL of ethyl acetate. 30 mL of deionized water was slowly added under an ice-water bath, and the aqueous phase was extracted twice with ethyl acetate (2 × 20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to obtain a crude product. The crude product was purified by reverse phase column chromatography (ACN:H2O = 70%) to obtain 1.5 g of compound Int-N-2 with a yield of 76% and a purity of 92%.

[0664] MS m / z(ESI):806.2[M+H2O].

[0665] Step 2: Synthesis of compound Int-N-4

[0666] KOH (1.92 g, 34.24 mmol) was added to a 25 mL single-necked flask at room temperature, and DMSO (anhydrous, 5 ml) was added and stirred evenly; compound Int-N-3 (1.67 g, 11.41 mmol) was added and stirred at room temperature for 15 minutes; compound Int-N-2 (1.5 g, 1.9 mmol) and KI (157 mg, 0.95 mmol) were added and stirred for 10 minutes; the temperature was raised to an external temperature of 75°C and the reaction was carried out for 6 hours; the mixture was cooled to room temperature, diluted with 20 mL of ethyl acetate, and 30 ml of deionized water was slowly added under an ice-water bath. The aqueous phase was extracted twice with ethyl acetate (2×20 ml), the organic phases were combined, washed with saturated brine (20 ml), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product; the crude product was purified by reverse phase column (ACN:H2O=90%) to obtain 1.5 g of compound Int-N-4 with a yield of 85% and a purity of 90%.

[0667] MS m / z(ESI):936.6[M+H2O].

[0668] Step 3: Synthesis of compound Int-N-5

[0669] Compound Int-N-4 (1.5 g, 1.63 mmol) was dissolved in anhydrous methanol (15 mL), and hydrochloric acid (0.4 mL, 5.0 mmol, 12 M) was added. The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated to obtain 1.1 g of crude compound Int-N-5, which was used directly in the next step.

[0670] MS m / z(ESI):667.4[M+H] + .

[0671] Step 4: Synthesis of compound Int-N-6

[0672] Under an ice-water bath, the crude compound Int-N-5 (1.1 g, 1.63 mmol) was dissolved in pyridine (20 mL), and p-toluenesulfonyl chloride (6.29 g, 33.0 mmol) was added. The reaction was stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated and the crude product was purified via a reverse-phase column (ACN:H2O = 90%) to obtain 0.75 g of compound Int-N-6, with a yield of 40% and a purity of 95%.

[0673] MS m / z(ESI):1129.5[M+H] + .

[0674] Step 5: Synthesis of compound Int-N-7

[0675] Compound Int-N-6 (0.75 g, 0.67 mmol) was dissolved in ethanolic methylamine (5 mL, approximately 30%) and stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated, and the concentrate was dissolved in DCM (10 mL). Boc2O (2.47 g, 11.33 mmol) and triethylamine (2.29 g, 22.66 mmol) were added, stirred overnight, and then concentrated directly. The crude product was purified via reverse phase column chromatography (ACN:H2O = 95%) to obtain 0.2 g of compound Int-N-7, with a yield of 35% and a purity of 94%.

[0676] MS m / z(ESI):1023.6[M+H2O].

[0677] Step 6: Synthesis of compound Int-N-8

[0678] Compound Int-N-7 ​​(0.2 g, 0.198 mmol) and 20% Pd(OH)2 / C (0.1 g) were added to a 50 mL single-necked flask, followed by the addition of MeOH (10 mL) and stirring at room temperature for 5 minutes. The mixture was then replaced three times under an atmospheric pressure of hydrogen, heated to 30°C, and reacted for 18 hours. The mixture was filtered through celite, and the filter cake was washed three times with methanol (5 mL × 3). The filtrate was directly concentrated to obtain 0.182 g of crude compound Int-N-8, which was used directly in the next step.

[0679] MS m / z(ESI):933.6[M+18].

[0680] Step 7: Synthesis of compound Int-N-9

[0681] Compound Int-N-8 (0.182 g, 0.198 mmol) was dissolved in anhydrous THF (5 mL), cooled to 0°C in an ice-water bath, and stirred; potassium tert-butoxide (0.5 mL, 0.5 mmol, 1 M in THF) was added, then the mixture was warmed to room temperature and stirred for one hour; the mixture was cooled in an ice-water bath again, 3-bromopropyne (70.9 mg, 0.595 mmol) was added dropwise, and the mixture was slowly warmed to room temperature and reacted for two hours; the mixture was cooled in an ice-water bath, quenched with saturated aqueous ammonium chloride (10 mL), and then extracted with EtOAc (10 mL×3). The organic phases were combined, washed with water (10 mL) and saturated brine (10 mL) in sequence, dried over anhydrous sodium sulfate (5 g), filtered, and dried under reduced pressure. The residue was purified by reverse phase column chromatography (ACN:0.1% TFA in H2O=65%-75%) to obtain 0.15 g of compound Int-N-9, yield: 79%.

[0682] MS m / z(ESI):971.6[M+H2O].

[0683] Step 8: Synthesis of compound Int-N-10

[0684] Compound Int-N-9 (0.15 g, 0.157 mmol) was dissolved in DCM (1.5 mL), cooled in an ice-water bath, stirred, and then TFA (1.5 mL) was added dropwise. The reaction was allowed to warm to room temperature and reacted for two hours. The reaction was stopped and concentrated directly. The residue was washed twice with DCM (5 mL x 2) and then pumped dry with an oil pump to obtain the crude compound Int-N-10, which was used directly in the next step.

[0685] MS m / z(ESI):654.3[M+H] + .

[0686] Step 9: Synthesis of compound Int-N-11

[0687] Compound Int-N-10 (103 mg, 0.157 mmol) was added to a single-necked flask, and DCM (2 mL) and DMF (2 mL) were added at room temperature. DIEA (0.41 g, 3.15 mmol) was added and stirred at room temperature for 5 minutes. Compound Int-A-7 (0.355 g, 0.472 mmol) was added in batches, replaced three times under nitrogen protection, and then heated to 49 ° C for 10 hours. The reaction was stopped and concentrated directly. The residue was dissolved in H2O (10 mL) and extracted with EA (10 mL × 2). The organic phases were combined, concentrated under reduced pressure, and then purified by reverse phase column (ACN / H2O, 90-93% product) to obtain 0.2 g of compound Int-N-11, yield: 50.95%.

[0688] MS m / z(ESI):831.4[M / 3+H] + .

[0689] Step 10: Synthesis of compound Int-N-12

[0690] Compound Int-N-11 (0.2 g, 0.083 mmol) was dissolved in MeOH (1.5 mL) and THF (1.5 mL), cooled in an ice-water bath, and H2O (1.5 mL) was added, followed by lithium hydroxide monohydrate (25 mg, 0.66 mmol). The reaction was allowed to warm to room temperature and stirred for 3 hours. A cation exchange resin was added until the pH was 5-6, followed by filtration. The filter cake was washed with MeOH (3 mL x 2), and the filtrate was concentrated to obtain 0.19 g of crude compound Int-N-12, which was directly used in the next step.

[0691] MS m / z(ESI):817.3[M / 3+H] + .

[0692] Step 11: Synthesis of Compound Int-N-13

[0693] Compound Int-N-12 (0.19 g, 0.08 mmol) was dissolved in DCM (1.8 mL), and TFA (1.8 mL) was added under ice-water cooling. The reaction was then warmed to room temperature and stirred for 2 hours; the mixture was directly concentrated, and the residue was washed twice with dichloromethane (3 mL), then dissolved in ACN (3 mL) and H2O (3 mL) and stirred at room temperature for 3 hours; the mixture was directly concentrated and purified by reverse phase column (ACN / 0.1% TFA in H2O, 40% product) to obtain 90 mg of the TFA salt of compound Int-N-13, with a yield of 46%.

[0694] MS m / z(ESI):577.1[M / 3+H] + .

[0695] Step 12: Synthesis of Compound Int-N

[0696] Under nitrogen protection, compound Int-N-13 (TFA salt, 80 mg, 0.035 mmol) and compound Int-L1-3 (140 mg, 0.35 mmol) were dissolved in NMP (1 mL), and copper tetraacetonitrile hexafluorophosphate (65 mg, 0.174 mmol) was added at room temperature, followed by reaction at room temperature for 20 minutes; the product was directly sent for preparative purification by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain 12.5 mg of compound Int-N TFA salt, yield: 10%, purity: 90.8%.

[0697] 1 H NMR (400MHz, D2O): δ7.98(s,1H),7.89-7.83(m,1H),7.01-6.97(m,1H),5.80(s,3H),4.91-4.85(m,5H),4.60-4.46(m,10H),4.34( d,5H),4.12-3.98(m,8H),3.88-3.81(m,6H),3.62-3.50(m,44H),3.45-3.29(m,10H),2.93-2.88(m,6H),2.81(s,6H),1.88(s,9H).

[0698] MS m / z(ESI):711.9[M / 3+H] + .

[0699] Example 22 Synthesis of Intermediate Int-O

[0700] Step 1: Synthesis of compound Int-O-1

[0701] KOH (0.96 g, 17.12 mmol) was added to a 25 mL single-necked bottle at room temperature, and DMSO (anhydrous, 5 ml) was added and stirred evenly; compound Int-N-3 (purchased from Leyan, 1.32 g, 9.0 mmol) was added and stirred at room temperature for 15 minutes; compound Int-M-1 (1.18 g, 2.0 mmol, prepared by a known method, Journal of Organic Chemistry) was added. Chemistry, 2012, vol. 77, # 20, p. 8879–8887) and KI (166 mg, 0.5 mmol) were stirred for 10 minutes; the temperature was raised to 75°C for reaction for 6 hours; the mixture was cooled to room temperature, diluted with 20 mL of ethyl acetate, and 20 mL of deionized water was slowly added under an ice-water bath. The aqueous phase was extracted twice with ethyl acetate (2×20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product; the crude product was purified by reverse phase column chromatography (ACN:H2O=90%) to obtain 1.0 g of compound Int-O-1 with a yield of 67% and a purity of 95%.

[0702] MS m / z(ESI):804.5[M+H2O].

[0703] Step 2: Synthesis of compound Int-O-2

[0704] Compound Int-O-1 (1.0 g, 1.27 mmol) was dissolved in anhydrous methanol (10 mL), and hydrochloric acid (0.53 mL, 6.4 mmol, 12 M) was added. The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated to obtain 0.9 g of crude compound Int-O-2, which was used directly in the next step.

[0705] MS m / z(ESI):535.3[M+H] + .

[0706] Step 3: Synthesis of compound Int-O-3

[0707] Under an ice-water bath, the crude compound Int-O-2 (0.9 g, 1.27 mmol) was dissolved in pyridine (15 mL) and p-toluenesulfonyl chloride (4.2 g, 22.0 mmol) was added. The reaction was stirred at room temperature overnight. After completion of the reaction, the mixture was concentrated and the crude product was purified via a reverse-phase column (ACN:H2O = 90%) to obtain 0.8 g of the compound Int-O-3 with a yield of 63% and a purity of 95%.

[0708] MS m / z(ESI):997.5[M+H] + .

[0709] Step 4: Synthesis of compound Int-O-4

[0710] Compound Int-O-3 (0.8 g, 0.8 mmol) was dissolved in ethanolic methylamine (8 mL, approximately 30%) and stirred at room temperature overnight. After the reaction, the mixture was concentrated and the concentrate was dissolved in DCM (10 mL). Boc2O (2.18 g, 10.0 mmol) and triethylamine (2.02 g, 20.0 mmol) were added, stirred overnight, and then concentrated directly. The crude product was purified via reverse-phase column chromatography (ACN:H2O = 95%) to obtain 0.5 g of compound Int-O-4, with a yield of 72% and a purity of 95%.

[0711] MS m / z(ESI):891.6[M+H2O].

[0712] Step 5: Synthesis of compound Int-O-5

[0713] Compound Int-O-4 (0.255 g, 0.291 mmol) was dissolved in 10 mL of MeOH and 20% palladium hydroxide on carbon (0.255 g) was added. The air in the system was replaced with hydrogen four times, and then the reaction was carried out at room temperature under a hydrogen atmosphere for ten hours. After the reaction was completed, the mixture was filtered and concentrated to obtain 0.22 g of crude compound Int-O-5, which was used directly in the next step. Yield: 96.1%

[0714] MS m / z(ESI):806.48[M+Na].

[0715] Step 6: Synthesis of compound Int-O-6

[0716] Compound Int-O-5 (0.2 g, 0.28 mmol) was dissolved in dry tetrahydrofuran (4 mL), and a solution of potassium tert-butoxide in THF (0.56 mL, 0.56 mmol) was added under ice-water cooling. After the addition, the reaction was stirred at room temperature for one hour. The mixture was then cooled in an ice-water bath, and propargyl bromide (0.1 g, 0.84 mmol) was added. After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction mixture was cooled in an ice-water bath and quenched with saturated ammonium chloride aqueous solution. The mixture was extracted with ethyl acetate, dried, concentrated, and purified by column chromatography (water:acetonitrile = 35%) to afford 0.174 g of compound Int-O-6, in a 75% yield.

[0717] MS m / z(ESI):844.53[M+Na].

[0718] Step 7: Synthesis of compound Int-O-7

[0719] Compound Int-O-6 (174 mg, 0.211 mmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (2 mL) was added under ice-water cooling. The reaction was stirred for 1 hour while naturally warming. After the reaction was complete, the mixture was concentrated to afford 147 mg of crude compound Int-O-7, which was used directly in the next step.

[0720] MS m / z(ESI):522.4[M+H] + .

[0721] Step 8: Synthesis of compound Int-O-8

[0722] To a solution of compound Int-O-7 (147 mg, 0.295 mmol) in N,N-dimethylformamide (4 mL) and dichloromethane (4 mL) was added N,N-diisopropylethylamine (728 mg, 5.64 mmol) and stirred at room temperature for 5 min. Compound Int-A-7 (635 mg, 0.845 mmol) was then added portionwise, and the mixture was heated to 49°C and stirred overnight. The reaction solution was concentrated at room temperature to remove dichloromethane and N,N-dimethylformamide, and purified by column chromatography (acetonitrile:water = 95%) to afford 274 mg of compound Int-O-8, a yield of 41%.

[0723] MS m / z(ESI):787.2[M / 3+H] + .

[0724] Step 9: Synthesis of compound Int-O-9

[0725] To a solution of compound Int-O-8 (274 mg, 0.116 mmol) in methanol (3 mL) and tetrahydrofuran (3 mL) was added a solution of lithium hydroxide monohydrate (38 mg, 0.928 mmol) in water (3 mL) under an ice-water bath. After the reaction was allowed to warm naturally, the reaction solution was adjusted to pH 5-6 using ion exchange resin IRN77. The filtered mother liquor was concentrated to afford 243 mg of compound Int-O-9, with a yield of 90%.

[0726] MS m / z(ESI):773.3[M / 3+H] + .

[0727] Step 10: Synthesis of compound Int-O-10

[0728] Under an ice-water bath, trifluoroacetic acid (2 mL) was added to a solution of compound Int-O-9 (243 mg, 0.104 mmol) in dichloromethane (2 mL). The reaction was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure, followed by addition of acetonitrile (2 mL) and water (1 mL), and stirred at room temperature for 3 hours. The reaction solution was concentrated and purified by column chromatography (0.1% TFA in H2O: acetonitrile = 75%) to obtain 213 mg of the TFA salt of compound Int-O-10, with a yield of 93.75%.

[0729] MS m / z(ESI):573.1[M / 3+H] + .

[0730] Step 11: Synthesis of compound Int-O

[0731] To a solution of compound Int-O-10 (TFA salt, 200 mg, 0.092 mmol) in N-methylpyrrolidone (0.5 mL) were added copper tetraacetonitrile hexafluorophosphate (103 mg, 0.276 mmol) and compound Int-L1-3 (149 mg, 0.369 mmol) in sequence. The reaction was stirred at room temperature for 20 minutes. The reaction solution was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient ratio: phase B 5%-95%) to obtain 38 mg of the TFA salt of compound Int-O in a yield of 20.57%.

[0732] 1 H NMR (400MHz, D2O): δ7.99(s,1H),7.77(t,1H),7.01-6.97(m,1H),5.66(s,3H),4.89-4.83(m,5H),4.59-4.31(m,13H),4.06(d,9H),3. 88-3.81(m,6H),3.62-3.42(m,42H),3.41-3.27(m,12H),3.22-2.97(m,3H),2.93-2.88(m,8H),2.81(s,6H),2.62(s,2H),1.87(s,9H).

[0733] MS m / z(ESI):667.8[M / 3+H] + .

[0734] Example 23 Synthesis of Intermediate Int-P

[0735] Step 1: Synthesis of compound Int-P-1

[0736] Compound Int-C-4 (1 g, 2.76 mmol) was dissolved in methanol (10 mL). N-tert-Butyloxycarbonyl-(methylamino)acetaldehyde (2.87 g, 16.55 mmol, purchased from Leyan) and 1 drop of acetic acid were added and stirred at 20°C for 1 hour. Sodium triacetoxyborohydride (2.92 g, 13.79 mmol) was added under ice-cold water, and the temperature was naturally raised to 20°C for 16 hours. The solvent was evaporated, and the residue was stirred with water and dichloromethane. The layers were separated, and the organic phase was concentrated to dryness and purified by column chromatography (methanol:dichloromethane = 7%) to obtain the product. The product was collected and dried to give 1.9 g of compound Int-P-1, with a yield of 82.57%.

[0737] MS (ESI): m / z 835.6[M+1] + .

[0738] Step 2: Synthesis of compound Int-P-2

[0739] Compound Int-P-1 (0.52 g, 623.43 μmol) was dissolved in dichloromethane (2.5 mL) and stirred. A 4.0 M solution of hydrogen chloride in dioxane (5 mL) was added under ice-cooling and stirred for 1-2 hours. The solvent was evaporated to dryness to obtain 330 mg of compound Int-P-2 (100% yield).

[0740] MS (ESI): m / z 535.4 [M+1] + .

[0741] Step 3: Synthesis of compound Int-P-3

[0742] Compounds Int-P-2 (0.33 g, 623.43 μmol) and Int-A-7 (1.82 g, 1.37 mmol) were dissolved in N,N-dimethylformamide (4 mL). Diisopropylethylamine (1.21 g, 9.35 mmol) was added under ice-cold water, and the mixture was allowed to warm to room temperature and react for 16 hours. The reaction mixture was directly applied to a reverse-phase column and purified by column chromatography (methanol:water = 100%) to obtain the product, which was then collected and dried to obtain 0.76 g of the TFA salt of compound Int-P-3 (yield 51.4%).

[0743] MS (ESI): m / z 1186.3 ([M+1] / 2) + .

[0744] Step 4: Synthesis of compound Int-P-4

[0745] Compound Int-P-3 (0.76 g, 320.46 μmol) was dissolved in methanol (6 mL) and water (6 mL). Lithium hydroxide monohydrate (80.69 mg, 1.92 mmol) was added under ice-cold water, and the mixture was allowed to warm to room temperature for 3 hours. The reaction mixture was then cooled with ice-cold water and the pH was adjusted to 5-6 with acidic resin. The mixture was filtered and dried to give 0.74 g of compound Int-P-4 (99% yield).

[0746] MS (ESI): m / z 1165.1 ([M+1] / 2) + .

[0747] Step 5: Synthesis of compound Int-P-5

[0748] Compound Int-P-4 (0.74 g, 317.66 μmol) was dissolved in dichloromethane (4 mL). Trifluoroacetic acid (3.5 mL) was added under ice-cold conditions and the mixture was allowed to warm to room temperature for 3 hours. The solvent was then dried and dissolved in water (4 mL). Reverse phase preparation (acetonitrile: 0.1% TFA in H2O = 25%) was performed to obtain 0.15 g of the TFA salt of compound Int-P-5, in a yield of 29.35%.

[0749] MS (ESI): m / z 804.9 ([M+1] / 2) + .

[0750] Step 6: Synthesis of compound Int-P

[0751] To a solution of compound Int-P-5 (TFA salt, 143 mg, 0.064 mmol) in N-methylpyrrolidone (1.0 mL) were added copper tetraacetonitrile hexafluorophosphate (71.8 mg, 0.193 mmol) and compound Int-L1-3 (155 mg, 0.385 mmol) in sequence. The reaction was stirred at room temperature for 20 minutes. The reaction solution was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient: phase B 5%-95%) to obtain 80 mg of the TFA salt of compound Int-P in a yield of 48.2%.

[0752] 1H NMR (400MHz, D2O): δ7.98(s,1H),7.89-7.83(m,1H),7.01-6.97(m,1H),5.88(d,3H),4.99-4.84(m,3H),4.6 2-4.32(m,14H),4.15-3.99(m,7H),3.89-3.74(m,10H),3.65-3.40(m,50H),2.93-2.82(m,9H),1.89(s,9H).

[0753] MS m / z(ESI):671.8[M / 3+H] + .

[0754] Example 24 Synthesis of Intermediate Int-I1

[0755] To a solution of compound Int-I (TFA salt, 182 mg, 0.0741 mmol) in N-methylpyrrolidone (1.5 mL) were added copper tetraacetonitrile hexafluorophosphate (83 mg, 0.222 mmol) and compound Int-L1-3 (179 mg, 0.444 mmol) in sequence. The reaction was stirred at room temperature for 20 minutes. The reaction solution was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient: phase B 5%-95%) to obtain 70 mg of the TFA salt of compound Int-I1 in a yield of 34.2%.

[0756] 1 H NMR(400MHz,D2O): δ7.99(s,1H),7.89-7.83(m,1H),7.01-6.98(m,1H),5.80(d,3H),4.90-4.86(m,3H),4.59-4.34(m,10H), 4.14-4.05(m,6H),3.88-3.74(m,10H),3.63-3.41(m,64H),3.21-3.09(m,3H),2.93-2.85(m,6H),2.81(s,6H),1.89(s,9H).

[0757] MS m / z(ESI):730.9[M / 3+H] + .

[0758] Example 25 Synthesis of Intermediate Int-G1

[0759] To a solution of compound Int-G (TFA salt, 240 mg, 0.089 mmol) in N-methylpyrrolidone (1.0 mL) were added copper tetraacetonitrile hexafluorophosphate (109 mg, 0.293 mmol) and compound 1-3 (237 mg, 0.586 mmol) in sequence. The reaction was stirred at room temperature for 20 minutes. The reaction solution was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient: phase B 5%-95%) to obtain 100 mg of the TFA salt of compound Int-G1 in a yield of 37.1%.

[0760] 1 H NMR (400MHz, D2O): δ7.99(s,1H),7.89-7.83(m,1H),7.01-6.99(m,1H),5.84(d,3H),4.91-4.87(m,3H),4.61-4.47(m,7H),4.38-4.3 5(m,3H),4.14-3.98(m,7H),3.88-3.78(m,16H),3.64-3.41(m,76H),3.21-3.11(m,4H),2.93-2.83(m,6H),2.81(s,6H),1.89(s,9H).

[0761] MS m / z(ESI):804.0[M / 3+H] + .

[0762] Example 26 Synthesis of Intermediate Int-D1

[0763] To a solution of compound Int-D (TFA salt, 260 mg, 0.139 mmol) in N-methylpyrrolidone (2.5 mL) were added copper tetraacetonitrile hexafluorophosphate (156 mg, 0.419 mmol) and compound Int-L1-3 (339 mg, 0.838 mmol) in sequence. The reaction was stirred at room temperature for 20 minutes. The reaction solution was purified by HPLC (Waters XBridge, 19*150 mm, 5 μm; mobile phase A: 0.05% TFA aqueous solution, mobile phase B: acetonitrile, gradient: phase B 5%-95%) to obtain 210 mg of the TFA salt of compound Int-D1 in a yield of 53.1%.

[0764] 1H NMR (400MHz, D2O): δ7.99(s,1H),7.89-7.83(m,1H),7.01-6.98(m,1H),5.91(d,3H),4.91-4.87(m,3H),4.59-4.48(m,6H),4.37-4.3 6(m,3H),4.13-3.97(m,6H),3.88-3.78(m,12H),3.64-3.41(m,70H),3.20-3.09(m,4H),2.93-2.87(m,6H),2.80(s,6H),1.88(s,9H).

[0765] MS m / z(ESI):759.9[M / 3+H] + .

[0766] Example 27 Preparation of Conjugate 1-I-E2

[0767] Preparation of 20 mM NaOAc / HOAc pH = 5.0: Dissolve 3.28 g of NaOAc in 1.8 L of water for injection, adjust the pH to 5.0 with concentrated HOAc, and adjust the volume to 2.0 L. Filter using a 20 μM membrane filtration system.

[0768] Preparation of 1.0M NaHCO3 / Na2CO3 pH 9.5: Phase A: 50mL 1.0M NaHCO3 (4.2g), Phase B: 50mL 1.0M Na2CO3 (5.3g). Add Phase B to Phase A and adjust the pH to 9.5 for later use.

[0769] Procedure: To a 2 mL reaction tube, add protein E2 (3.0 mg, 0.05 μmol, 103.5 μL, the sequence of which is shown in SEQ ID NO: 8) and 20 mM NaOAc / HOAc pH=5.0 buffer (46.5 μL). Adjust the pH to between 9.0 and 9.5 (approximately pH=9.23) with 1.0 M NaHCO3 / Na2CO3 pH=9.5 (6 μL). Add Int-I1 (593.03 μg, 0.27 μmol, 5 eq) in DMSO (9 μL), pH=8.83, and adjust the pH to between 9.0 and 9.5 (approximately pH=9.21) with 1.0 M NaHCO3 / Na2CO3 pH=9.5 (0.6 μL). Oscillate at 25°C for 1 hour. Every hour, 2 / 2 / 1 eq of Int-I1 was added, and the pH was adjusted to 9.0-9.5, pH = 9.35, with 1.0 M NaHCO3 / Na2CO3, pH = 9.5 (0.5 uL). The mixture was shaken at 25°C for 1 hour. This addition was repeated three times, resulting in a total of 10 eq of the small molecule. The reaction mixture was then ultrafiltered four times using 5.0% PBS buffer to obtain the coupled product 1-I-E2 (2.92 mg, 97.4% yield, 98.3% purity). Mass spectrometry analysis revealed a DAR of 4.36.

[0770] Example 28 Preparation of Conjugate 1-M-E2

[0771] Procedure: Prepared using the same method as in Example 27. Int-M was added three times at 1 / 1 / 1 eq every 1 h, for a total of 8 eq of the small molecule. Ultrafiltration afforded the coupled product 1-M-E2 (2.51 mg, 83.7% yield, 98.4% purity). Mass spectrometry analysis revealed a DAR of 4.48.

[0772] Example 29 Preparation of Conjugate 1-G-E2

[0773] Procedure: Prepared using the same method as in Example 27. Int-G1 was added twice, at 2 / 1 eq, every 1 h, for a total of 8 eq of the small molecule. Ultrafiltration afforded the coupled product, 1-G-E2 (2.98 mg, 99.5% yield, 98.6% purity). Mass spectrometry analysis revealed a DAR of 4.14.

[0774] Example 30 Preparation of Conjugate 1-P-E2

[0775] Procedure: Prepared using the same method as in Example 27. Int-P was added three times at 1 hour intervals (2 / 2 / 1 eq, respectively), for a total of 10 eq of the small molecule. Ultrafiltration afforded the coupled product, 1-P-E2 (2.95 mg, 98.4% yield, 98.4% purity). Mass spectrometry analysis revealed a DAR of 4.54.

[0776] Example 31 Preparation of Conjugate 1-L-E1

[0777] Procedure: Prepared using the same method as in Example 27, the protein Fc sequence is shown in SEQ ID NO: 1. Int-L1 was added three times at 1 / 1 / 1 eq intervals, for a total of 8 eq of the small molecule. Ultrafiltration yielded the coupled product 1-L-E1 (2.88 mg, 96.1% yield, 99.3% purity). Mass spectrometry analysis revealed a DAR of 4.46.

[0778] Example 32 Preparation of Conjugate 1-L-E3

[0779] Procedure: The same method as in Example 27 was used for preparation, using hIgG1 (heavy chain sequence shown in SEQ ID NO: 9, light chain sequence shown in SEQ ID NO: 10). Int-L1 was added three times at 1-hour intervals, 2 / 2 / 1 eq, for a total of 10 eq of the small molecule. After ultrafiltration, the coupling product 1-L-E3 (2.86 mg, 95.6% yield, 98.7% purity) was obtained. Mass spectrometry analysis revealed a DAR of 4.59.

[0780] Example 33 Preparation of Conjugate 1-L-E4

[0781] Procedure: The same method as in Example 27 was used for preparation, using human serum albumin (MCE, Catalog No. HY-P1956-100 mg). Int-L1 was added twice at 2 / 2 eq intervals every 1 h, for a total of 9 eq of the small molecule. Ultrafiltration yielded 80.1% purity. The coupled product, 1-L-E4, was obtained by filtration through a HiLoad Superdex 200 pg preparative SEC column (mobile phase: 1×PBS 5.0) to yield 0.97 mg, 32.3% yield, 95.6% purity. Mass spectrometry analysis revealed a DAR of 4.53.

[0782] Example 34 Preparation of Conjugate 1-O-E2

[0783] Procedure: Prepared using the same method as in Example 27. 2 / 2 eq of Int-O was added twice every 1 h, for a total of 9 eq of the small molecule. Ultrafiltration afforded the coupled product 1-O-E2 (2.65 mg, 88.6% yield, 98.3% purity). Mass spectrometry analysis revealed a DAR of 4.46.

[0784] Example 35 Preparation of Conjugate 1-N-E2

[0785] Procedure: Prepared using the same method as in Example 27. 2 eq of Int-N was added every 1 h, for a total of 7 eq of the small molecule. Ultrafiltration afforded the coupled product 1-N-E2 (2.94 mg, 98.2% yield, 98.6% purity). Mass spectrometry analysis revealed a DAR of 4.28.

[0786] Example 36 Preparation of Conjugate 1-L-E2

[0787] Procedure: Prepared using the same method as in Example 27. 1 / 1 eq of Int-L1 was added twice every 1 h, for a total of 7 eq of the small molecule. Ultrafiltration afforded the coupled product 1-L-E2 (2.40 mg, 80.6% yield, 98.8% purity). Mass spectrometry analysis revealed a DAR of 4.39.

[0788] Example 37 Preparation of Conjugate 1-D-E2

[0789] Procedure: Prepared using the same method as in Example 27. Int-D1 was added twice, at 1 / 1 eq, every 1 h, for a total of 7 eq of the small molecule. Ultrafiltration afforded the coupled product, 1-D-E2 (2.77 mg, 92.3% yield, 98.8% purity). Mass spectrometry analysis revealed a DAR of 4.28.

[0790] Example 38 Preparation of Conjugate 1-L-E2

[0791] Step 1: 1: Fc-azido (7.66 mg, yield 76.6%, purity 99.3%, Fc sequence shown in SEQ ID NO: 8) was prepared using the same method as in Example 13.

[0792] Step 2: To a 15 mL reaction tube, add Fc-azido (7.66 mg), compound Int-L (3.61 mg, 15.0 eq) in 1×PBS pH 6.3 (277.7 μL), and click reagent (428 μL). Shake at room temperature for 20 hours. After desalting with PBS (pH 5.0), the mixture was purified by passing through a HiLoad Superdex 200 pg preparative SEC column (mobile phase: 1×PBS 5.0). The collected solution was concentrated to yield conjugate 1-L-E2 (5.26 mg, 68.7% yield, 98.0% purity). Maldi ion analysis revealed a DAR value of 4.25.

[0793] Biological evaluation

[0794] The present disclosure is further described and explained below in conjunction with test examples, but these embodiments are not intended to limit the scope of the present disclosure.

[0795] Test Example 1: Cellular-level detection of conjugate molecule cytotoxicity and anti-influenza virus function

[0796] 1. Test sample

[0797] Test compounds (Conjugate 1-D-E1, Conjugate 1-L-E1): The starting concentration of the test was 1 μM, and the compounds were diluted to 9 concentrations in a 3.16-fold gradient.

[0798] Reference drug 1: Zanamivir (purchased from Shanghai Bidex, batch number 139110-90-8), the initial test concentration was 100 μM, and a total of 9 concentrations were diluted in a 3.16-fold gradient.

[0799] Reference drug 2: Conjugate C3-E1 (prepared according to the method of Example 13, the structure of the intermediate control compound used is shown below, prepared with reference to the synthesis method of Example 145.Int-83 in WO2021046549A1), the test starting concentration was 1 μM, and a total of 9 concentrations were diluted in a 3.16-fold gradient.

[0800] 2. Experimental instruments and materials

[0801] 3. Test methods

[0802] 1) Preparation of detection reagents:

[0803] MDCK cell culture medium: DMEM + 10% FBS + 1% Penicillin-Streptomycin

[0804] 2g / ml Trypsin: Dissolve 10g Trypsin powder in 5ml DMEM medium

[0805] Pre-infection cell culture medium: OptiPRO TM SFM+1% Penicillin-Streptomycin

[0806] Cell culture medium for infection: OptiPRO TM SFM + 2ug / ml Trypsin

[0807] 2) Detection method:

[0808] The cultured MDCK cells were resuspended in OptiPRO TM SFM medium, 5 × 10 3 Per 100 μl OptiPRO TM Each well of a 96-well plate was seeded with SFM + 1% Penicillin-Streptomycin and cultured overnight in a 37°C incubator (cell culture incubator model: ESCO CCL-170B-8). TM Dilute to the specified concentration in SFM + 2ug / ml Trypsin, and add 50μl per well to the wells where cells were pre-plated. Incubate at 37℃ for 2 hours, and then TM Dilute influenza virus (Influenza H1N1A / PR / 8 / 34) in SFM + 2ug / ml Trypsin and add 50μl per well to the wells containing cells and compound. Incubate the mixed 96-well plate at 37°C for 5 days. After 5 days, add 20μl of CCK-8 solution to each well. Incubate in a 37°C incubator for 2 hours, and then measure absorbance at 450nm.

[0809] 4. Test results

[0810] Table 1 EC values ​​of conjugates / compounds in MDCK cells 50 With CC 50

[0811] Test Example 2: Cellular-level detection of the anti-Influenza H1N1 A / PR / 8 / 34 influenza virus function of the conjugate molecule

[0812] 1. Test sample

[0813] Test compounds (conjugate 1-D-E2, conjugate 1-L-E2): The starting concentration of the test was 1 μM, and the compounds were diluted to 9 concentrations in a 3.16-fold gradient.

[0814] Reference drug 1: Zanamivir (purchased from Shanghai Bidex, batch number 139110-90-8), the initial test concentration was 100 μM, and a total of 9 concentrations were diluted in a 3.16-fold gradient.

[0815] Reference drug 2: conjugate C3-E1, the starting concentration of the test was 1 μM, and 9 concentrations were diluted in a 3.16-fold gradient.

[0816] 2. Experimental instruments and materials

[0817] 3. Test methods

[0818] 1) Preparation of detection reagents:

[0819] MDCK cell culture medium: DMEM + 10% FBS + 1% Penicillin-Streptomycin

[0820] 2g / ml Trypsin: Dissolve 10g Trypsin powder in 5ml DMEM medium

[0821] Pre-infection cell culture medium: OptiPRO TM SFM+1% Penicillin-Streptomycin

[0822] Cell culture medium for infection: OptiPRO TM SFM + 2ug / ml Trypsin

[0823] 2) Detection method:

[0824] The cultured MDCK cells were resuspended in OptiPRO TM SFM medium, 5 × 10 3 Per 100 μl OptiPRO TM Each well of a 96-well plate was seeded with SFM + 1% Penicillin-Streptomycin and cultured overnight in a 37°C incubator (cell culture incubator model: ESCO CCL-170B-8). TM Dilute to the specified concentration in SFM + 2ug / ml Trypsin, and add 50μl per well to the wells where cells were pre-plated. Incubate at 37℃ for 2 hours, and then TMDilute influenza virus (Influenza H1N1A / PR / 8 / 34) in SFM + 2ug / ml Trypsin and add 50μl per well to the wells containing cells and compound. Incubate the mixed 96-well plate at 37°C for 5 days. After 5 days, add 20μl of CCK-8 solution to each well. Incubate in a 37°C incubator for 2 hours, and then measure absorbance at 450nm.

[0825] 4. Test results

[0826] Table 2 Conjugates / compounds against influenza H1N1 A / PR / 8 / 34 virus EC 50

[0827] Test Example 3: Cellular Detection of the Anti-Influenza B / Lee Virus Function of the Conjugate Molecule

[0828] 1. Test sample

[0829] Test compounds (conjugate 1-D-E2, conjugate 1-L-E2): The starting concentration of the test was 1 μM, and the compounds were diluted to 9 concentrations in a 3.16-fold gradient.

[0830] Reference drug: Zanamivir (purchased from Shanghai Bidex, batch number 139110-90-8), the initial test concentration was 100 μM, and the test was diluted in a 3.16-fold gradient to a total of 9 concentrations.

[0831] 2. Experimental instruments and materials

[0832] 3. Test methods

[0833] 1) Preparation of detection reagents:

[0834] MDCK cell culture medium: DMEM + 10% FBS + 1% Penicillin-Streptomycin

[0835] 2g / ml Trypsin: Dissolve 10g Trypsin powder in 5ml DMEM medium

[0836] Pre-infection cell culture medium: OptiPRO TM SFM+1% Penicillin-Streptomycin

[0837] Cell culture medium for infection: OptiPRO TM SFM + 2ug / ml Trypsin

[0838] 2) Detection method:

[0839] The cultured MDCK cells were resuspended in OptiPRO TM SFM medium, 5 × 10 3 Per 100 μl OptiPRO TM Each well of a 96-well plate was seeded with SFM + 1% Penicillin-Streptomycin and cultured overnight in a 37°C incubator (cell culture incubator model: ESCO CCL-170B-8). TM Dilute to the specified concentration in SFM + 2ug / ml Trypsin, and add 50μl per well to the wells where cells were pre-plated. Incubate at 37℃ for 2 hours, and then TM Dilute influenza virus (Influenza B / Lee) in SFM + 2ug / ml Trypsin and add 50μl per well to the wells containing cells and compound. Incubate the mixed 96-well plate at 37°C for 5 days. After 5 days, add 20μl of CCK-8 solution to each well. Incubate in a 37°C incubator for 2 hours, and then measure absorbance at 450nm.

[0840] 4. Test results

[0841] Table 3 Conjugates / compounds against Influenza B / Lee virus EC 50

[0842] Test Example 4: Cellular Detection of the Anti-Influenza H1N1 A / PR / 8 / 34 Virus Function of the Conjugate Molecule

[0843] 1. Test sample

[0844] Test compounds (conjugate 1-L-E2, conjugate 1-L-E3, conjugate 1-L-E4): The starting concentration of the test was 10 μM, and 12 concentrations were diluted in a 3-fold or 2-fold gradient.

[0845] Reference drug 1: Zanamivir (purchased from Shanghai Bidex, batch number 139110-90-8), the initial test concentration was 300 μM, and 12 concentrations were diluted in a 3-fold or 2-fold gradient.

[0846] 2. Experimental instruments and materials

[0847] 3. Test methods

[0848] 1) Preparation of detection reagents:

[0849] MDCK cell culture medium: DMEM + 10% FBS + 1% Penicillin-Streptomycin

[0850] 2g / ml Trypsin: Dissolve 10g Trypsin powder in 5ml DMEM medium

[0851] Pre-infection cell culture medium: OptiPRO TM SFM+1% Penicillin-Streptomycin

[0852] Cell culture medium for infection: OptiPRO TM SFM + 2ug / ml Trypsin

[0853] 2) Detection method:

[0854] The cultured MDCK cells were resuspended in OptiPRO TM SFM medium, 5 × 10 3 Per 100 μl OptiPRO TM Each well of a 96-well plate was seeded with SFM + 1% Penicillin-Streptomycin and cultured overnight in a 37°C incubator (cell culture incubator model: Haier, HCB-168). TM Dilute to the specified concentration in SFM + 2ug / ml Trypsin, and add 50μl per well to the wells where cells were pre-plated. Incubate at 37℃ for 2 hours, and then TM Influenza virus (Influenza H1N1 A / PR / 8 / 34) was diluted in SFM + 2 μg / ml Trypsin and added to the wells containing cells and compound at a volume of 50 μl per well. The mixed 96-well plate was incubated at 37°C and the cells were observed daily. When the pathological changes reached 80% or more, cell viability was measured using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay Kit in a microplate reader, and the inhibition rate was calculated.

[0855] 4. Test results

[0856] Table 4 Conjugates / compounds against influenza H1N1 A / PR / 8 / 34 virus EC 50

[0857] Test Example 5: Cellular Detection of the Antiviral Function of the Combined Molecule against Influenza H1N1 A / PR / 8 / 34 Virus

[0858] 1. Test sample

[0859] Test compounds (conjugate 1-M-E2, conjugate 1-N-E2, conjugate 1-O-E2): The starting concentration of the test was 10 μM, and the compounds were diluted in 3- or 2-fold gradients to 12 concentrations.

[0860] Reference drug 1: Zanamivir (purchased from Shanghai Bidex, batch number 139110-90-8), the initial test concentration was 300 μM, and 12 concentrations were diluted in a 3-fold or 2-fold gradient.

[0861] 2. Experimental instruments and materials

[0862] 3. Test methods

[0863] 1) Preparation of detection reagents:

[0864] MDCK cell culture medium: DMEM + 10% FBS + 1% Penicillin-Streptomycin

[0865] 2g / ml Trypsin: Dissolve 10g Trypsin powder in 5ml DMEM medium

[0866] Pre-infection cell culture medium: OptiPRO TM SFM+1% Penicillin-Streptomycin

[0867] Cell culture medium for infection: OptiPRO TM SFM + 2ug / ml Trypsin

[0868] 2) Detection method:

[0869] The cultured MDCK cells were resuspended in OptiPRO TM SFM medium, 5 × 10 3 Per 100 μl OptiPRO TM Each well of a 96-well plate was seeded with SFM + 1% Penicillin-Streptomycin and cultured overnight in a 37°C incubator (cell culture incubator model: Haier, HCB-168). TM Dilute to the specified concentration in SFM + 2ug / ml Trypsin, and add 50μl per well to the wells where cells were pre-plated. Incubate at 37℃ for 2 hours, and then TMInfluenza virus (Influenza H1N1 A / PR / 8 / 34) was diluted in SFM + 2 μg / ml Trypsin and added to the wells containing cells and compound at a volume of 50 μl per well. The mixed 96-well plate was incubated at 37°C and the cells were observed daily. When the pathological changes reached 80% or more, cell viability was measured using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay Kit in a microplate reader, and the inhibition rate was calculated.

[0870] 4. Test results

[0871] Table 5 Conjugates / compounds against influenza H1N1 A / PR / 8 / 34 virus EC 50

[0872] Test Example 6: Pharmacokinetic study of different test articles administered by single intravenous injection to cynomolgus monkeys

[0873] 1. Test sample

[0874] Reference (conjugate C3-E1) and test product (conjugate 1-L-E2), the specific information of each test product is shown in Table 6.

[0875] Table 6. Sample information

[0876] Preparation method of drug delivery:

[0877] 1) Vehicle: PBS for injection ( Batch number: 2436336).

[0878] 2) Test Sample Preparation: Perform aseptic procedures in a biosafety cabinet and / or clean bench, using sterilized containers. Remove the test sample from the -80°C freezer and allow it to completely thaw. Then, dispense directly according to the test sample concentration or dilute with solvent to the desired dosing concentration. The actual dosage, concentration of the dosing preparation, and dosing volume for each group are shown in Table 7. The dosing preparation for each group should be prepared immediately upon completion and administered intravenously through a limb.

[0879] Table 7. Dosage, concentration, and volume of each group

[0880] 2. Experimental Animals

[0881] Cynomolgus macaques (common grade), two per group, half male and half female, aged 5.0–6.0 years, were obtained from Guangxi Fangchenggang Changchun Biotechnology Development Co., Ltd. and Hainan Jingang Biotechnology Co., Ltd., with certificate numbers: 0003029, 460012000000545, and 44818300000229. All experimental animals completed quarantine and were housed in individual cages at a temperature of 18–26°C, a humidity of 40–70%, and ≥8 air changes per hour using 100% fresh air. Animals underwent a 2–5 day acclimatization period before the experiment. Experimenters observed the animals within 2–3 days before dosing. Animals were randomly assigned to groups based on body weight on the day of the experiment.

[0882] 3. Test methods

[0883] Weigh before administration, calculate the dosage based on body weight, and administer the same dose to both groups via intravenous injection. Blood (1 mL / mouse / time point) was collected from the limb veins before administration and at 0.25 h, 2 h, 6 h, 24 h (D1), 72 h (D3), 120 h (D5), 168 h (D7), 240 h (D10), 336 h (D14), and 672 h (D28) after the start of administration. Whole blood was collected into tubes containing a coagulant and a separating gel coagulant. The blood was allowed to stand at room temperature for approximately 30 min before centrifugation. Within 1 hour after blood collection, the blood sample was allowed to agglutinate and then centrifuged at 4°C (2000 g, 10 minutes). The collected serum was stored at -80°C. Drug concentrations were measured using ELISA: Goat anti-human IgG Monkey ads UNLB antibody (Southern Biotech, Cat No. 2049-01) was added to a 96-well ELISA plate (Corning, Cat No. 9018) at a concentration of 1 μg / ml in a volume of 100 μL / well. The plate was incubated overnight at 4°C to coat the plate. After discarding the solution, the plate was washed five times with PBST (pH 7.4, 0.05% Tween-20) buffer. 200 μL of 4% bovine serum albumin solution was added to each well and incubated at room temperature for 2 hours for blocking. The blocking buffer was discarded, and the plate was washed four times with PBST buffer. 100 μL of the diluted sample was added to each well and incubated at room temperature for 1.5 hours. After incubation, the reaction solution was discarded, and the plate was washed four times with PBST. 100 μL of Goat Anti-Human IgG Monkey ads HRP detection antibody (Southern Biotech, Cat No. 2049-05) diluted 1:25k was added to each well and incubated for 1 hour at room temperature. After washing the plate four times with PBST, 100 μL of TMB (Sigma, Cat No. T0440) chromogenic substrate was added and incubated at room temperature for 10 minutes in the dark. The reaction was terminated with 1 M sulfuric acid, and the absorbance was read at 450 nm using a SpectraMax M5 microplate reader.

[0884] 4. Test results

[0885] The pharmacokinetic curves and parameters of each group of animals are shown in Figure 2 and Table 8 (data are expressed as Mean ± SEM). The test results show that after intravenous administration at the same dose, the AUC of the test conjugate 1-L-E2 group is last The concentration of the test conjugate 1-L-E2 group was 14466±123h*μg / mL, which was 1.7 times higher than that of the reference conjugate C3-E1 group (8691±1713h*μg / mL). 1 / 2The drug exposure and half-life of the 1-L-E2 conjugate in cynomolgus monkeys were significantly increased compared to the C3-E1 conjugate (687±79h), a two-fold increase compared to the reference conjugate C3-E1 (342±85h).

[0886] Table 8 PK parameters of different test products given to cynomolgus monkeys after single intravenous injection

Claims

1. A conjugate represented by formula (I), in, E is a protein or polypeptide; L is a linker that covalently links E to D; m is selected from 3, 4, 5, 6, 7, 8, 9 and 10; n is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any number between any two numbers); D is independently selected from the structure shown in formula (A), R1 is selected from -OH, -NH2 and -NHC(=NH)NHR5; R2 is selected from -CO2H, -P(=O)(OH)2 and -SO3H; R3 is -O- or -S-; R4 is selected from -COCH3, -COCF3 and -SO2CH3; R5 is selected from hydrogen, hydroxyl, thiol, nitro, cyano, -NR i R j 、-C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k 、C1-C 10 Alkyl, C1-C 10 Alkoxy, C2-C 10 Alkenyl and C2-C 10 Alkynyl, wherein the alkyl, alkoxy, alkenyl and alkynyl are optionally selected from C1-C6 alkyl, halogen, hydroxyl, thiol, -NR i R j , oxo, thio, -C(O)R k 、-C(O)OR k 、-S(O)R k 、-S(O)OR k 、-S(O)(O)R k 、-S(O)(O)OR k 、-C(S)R k , nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; R i , R j Each is independently selected from a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group and a C1-C6 alkoxy group; R k independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl, -NR i R j wherein the alkyl, alkoxy, and haloalkyl are optionally selected from C1-C6 alkyl, halogen, hydroxyl, mercapto, -NR i R j The invention may be substituted by one or more substituents selected from the group consisting of: 1-C6 alkyl group, 2-C6 alkyl group, 3-C6 alkyl group, 4-C6 alkyl group and 5-C6 alkyl group.

2. The conjugate according to claim 1, wherein R5 is selected from hydrogen, hydroxy, amino and -C(O)R k , R k Selected from hydrogen atom, C1-C6 alkyl, C1-C6 alkoxy and hydroxy, wherein the alkyl and alkoxy are optionally substituted by one or more substituents selected from halogen, hydroxy, oxo, thio, carboxyl, C1-C6 alkoxy, C1-C6 alkylthio, 6 to 10 membered aryl and 5 to 10 membered heteroaryl.

3. The conjugate according to claim 1, wherein D is selected from the structure shown in formula (A-1), 4. The conjugate according to any one of claims 1 to 3, wherein m is 3 or 4.

5. The conjugate according to any one of claims 1 to 4, wherein the linker comprises a core portion L2 and branch portions L1 and L3, the branch portion L1 is used to connect E to the core portion, and the branch portion L3 is used to connect D to the core portion.

6. The conjugate according to claim 5, wherein the core structure L2 is selected from in, y1 is an integer from 0 to 100, preferably an integer from 0 to 30, y2 is an integer from 0 to 10, preferably an integer from 0 to 6, more preferably 1 or 2, and Y b1 is selected from O or CH2, p1 and p2 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, or wherein z1 is an integer from 0 to 100, preferably an integer from 0 to 30, z2 is an integer from 0 to 10, preferably an integer from 0 to 6, more preferably 1 or 2, and Y b2 is selected from O or CH2, z3 is an integer from 0 to 100, preferably an integer from 0 to 30, z4 is an integer from 0 to 10, preferably an integer from 0 to 6, more preferably 1 or 2, Y b3 is selected from O or CH2, q1, q2, q3, q4 are each independently selected from an integer of 0-10, preferably an integer of 0-6; or 7. The conjugate according to claim 5 or 6, wherein L1 is selected from -G a1 -(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -, wherein xa1, xa3 are each independently selected from an integer of 0 to 100, preferably an integer of 0 to 30, xa2, xa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, Y a1 , Y a2 Each independently selected from O or CH2, G a1 For group G x , G a2 , G a3 Each independently selected from the group G x or does not exist, G x Selected from g is each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl.

8. The conjugate according to any one of claims 5 to 7, wherein L3 comprises a terminal group G connected to D c1 , the terminal group G c1 Selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl, preferably from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2-; Preferably L3 is -(CH2) wc1 -[Y c1 -CH2(CH2) xc2 ] xc1 -(CH2) wc2 -G c1 -, in, xc1 are each independently an integer from 0 to 100, preferably an integer from 0 to 30, more preferably an integer from 2 to 30, xc2 is each independently an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y c1 are each independently selected from O or CH2, preferably O, wc1 and wc2 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 0, 1, 2 or 3, G c1 Each independently selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl, preferably from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2; m is 3 or 4.

9. The conjugate according to any one of claims 1 to 8, wherein -L(-D) m -L1-L2(-L3-D) m ,in, L1 is a part connected to E, and L1 is selected from -G a1 -(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -,in, xa1 and xa3 are each independently selected from an integer from 0 to 100, preferably an integer from 0 to 30, xa2 and xa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, Y a1 , Y a2 are each independently selected from O or CH2, G a1 For group G x , G a2 , G a3 Each independently selected from the group G x or does not exist; L2 is selected from wherein y1 is an integer from 0 to 100, preferably an integer from 0 to 30, y2 is an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y b1 Selected from O or CH2, p1 and p2 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2; or wherein z1 is an integer from 0 to 100, preferably an integer from 0 to 30, z2 is an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y b2 is selected from O or CH2, z3 is an integer from 0 to 100, preferably an integer from 0 to 30, z4 is an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y b3 Selected from O or CH2, q1, q2, q3, q4 are each independently selected from an integer of 0-10, preferably an integer of 0-6; or L3 is -(CH2) wc1 -[Y c1 -CH2(CH2) xc2 ] xc1 -(CH2) wc2 -G c1 -,in, xc1 are each independently an integer from 0 to 100, preferably an integer from 0 to 30, more preferably an integer from 2 to 30, xc2 is each independently an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y c1 are each independently selected from O or CH2, wc1 and wc2 are each independently selected from an integer of 0-10, preferably an integer of 0-6, G c1 Each is independently selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl; G x Selected from g is each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl; m is 3 or 4.

10. The conjugate according to claim 8 or 9, wherein the number of atoms in the main chain of L3 is greater than 7, preferably greater than 8, more preferably greater than 9.

11. The conjugate according to any one of claims 1 to 10, wherein the protein or polypeptide is selected from an Fc domain, albumin or an albumin binding domain.

12. The conjugate according to any one of claims 1-11, wherein the Fc domain has enhanced effector function, preferably, the effector function is selected from: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cytotoxicity, phagocytosis, downregulation of cell surface receptors and B cell activation.

13. The conjugate according to any one of claims 1 to 12, wherein the Fc domain comprises an Fc region derived from IgG, preferably, the Fc region is selected from the Fc region of human IgG1, IgG2, IgG3, and IgG4; more preferably, the Fc region is human IgG1.

14. The conjugate according to any one of claims 1 to 13, which is selected from: in, L1 is selected from -G a1 -(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -, wherein xa1, xa3 are each independently selected from an integer of 0 to 100, preferably an integer of 0 to 30, xa2, xa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, Y a1 , Y a2 Each independently selected from O or CH2, G a1 Selected from group G x , G a2 , G a3 Each independently selected from the group G x or does not exist; G x Selected from g is each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl; G c1 Selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)- and -NR6(C=O)CH2-, wherein R6 is selected from hydrogen and C1-C6 alkyl; n is any value between 1 and 15. E as described in claim 1.

15. The conjugate according to any one of claims 1 to 13, which is selected from: in, xa5 are each independently selected from an integer between 2 and 8, G a1 Each independently selected from g is each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl; n is any value between 1 and 15. E as described in claim 1.

16. The conjugate according to any one of claims 1 to 15, wherein n is any value between 1 and 15, preferably any value between 2 and 12.

17. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

18. Use of the conjugate according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating a viral infection, wherein the viral infection is preferably caused by influenza virus or parainfluenza virus, and preferably the viral infection is influenza virus A, B or C or parainfluenza virus.

19. The compound shown below, L1'-L2(-L3-D) m , in, L1' is selected from G x '-(CH2) wa1 -[Y a1 -CH2(CH2) xa2 ] xa1 -(CH2) wa2 -G a2 -(CH2) wa3 -[Y a2 -CH2(CH2) xa4 ] xa3 -(CH2) wa4 -G a3 -,in, xa1 and xa3 are each independently selected from an integer from 0 to 100, preferably an integer from 0 to 30, xa2 and xa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, wa1, wa2, wa3, wa4 are each independently selected from an integer of 0-10, preferably an integer of 0-6, Y a1 , Y a2 Each independently selected from O or CH2; G x 'Each independently selected from G a2 , G a3 Each independently selected from: or does not exist; g is each independently selected from 0, 1, 2, 3, 4; R g are each independently selected from hydrogen or methyl; Ring H is a 5- to 10-membered heteroaryl group, Best Best choice R h are each independently selected from halogen; h is selected from 0, 1, 2, 3, 4, 5; h1 is independently selected from 0, 1, and 2; h2 is independently selected from 0, 1, 2, 3; h3 is independently selected from 0, 1, 2, 3, 4; h4 are each independently selected from 0, 1, 2, 3, 4, 5; L2 is selected from wherein y1 is an integer from 0 to 100, preferably an integer from 0 to 30, y2 is an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y b1 is selected from O or CH2, p1 and p2 are each independently selected from an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2; or wherein z1 is an integer from 0 to 100, preferably an integer from 0 to 30, z2 is an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y b2 is selected from O or CH2, z3 is an integer from 0 to 100, preferably an integer from 0 to 30, z4 is an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y b3 Selected from O or CH2, q1, q2, q3, q4 are each independently selected from an integer of 0-10, preferably an integer of 0-6; or L3 is -(CH2) wc1 -[Y c1 -CH2(CH2) xc2 ] xc1 -(CH2) wc2 -G c1 -,in, xc1 are each independently an integer from 0 to 100, preferably an integer from 0 to 30, xc2 is each independently an integer of 0-10, preferably an integer of 0-6, more preferably 1 or 2, Y c1 are each independently selected from O or CH2, wc1 and wc2 are each independently selected from an integer of 0-10, preferably an integer of 0-6, c1 Selected from -NR6(C=O)-, -NR6(C=S)-, -O(C=O)-, -(C=O)-, -NR6(C=O)CH2- or a chemical bond, wherein R6 is selected from hydrogen and C1-C6 alkyl; D is m is 3 or 4.

20. The compound according to claim 19, which is selected from: in, xa6 are each independently selected from an integer between 0 and 8, G x 'Each independently selected from 21. The compound according to claim 19 or 20, which is selected from: